Methods and compositions

ABSTRACT

The invention relates to a complex comprising a phage particle, said phage particle comprising
         (i) a polypeptide;   (ii) a nucleic acid encoding the polypeptide of (i);   (iii) a connector compound attached to said polypeptide   wherein said connector compound is attached to the polypeptide by at least three discrete covalent bonds. The invention also relates to libraries, and to methods for making complexes and to methods of screening using same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.13/973,297, filed 8 Aug. 2013, which is a continuation application ofU.S. Ser. No. 12/866,214, filed 4 Aug. 2010, now U.S. Pat. No.8,680,022, which is a 371(c)(1) filing of PCT/GB09/00301 filed 4 Feb.2009; which claims priority to GB0802079.4, filed 5 Feb. 2008 andGB0818399.8, filed 8 Oct. 2008.

FIELD OF THE INVENTION

The invention relates to the modification and constraint ofpolypeptides, in particular to genetically encoded polypeptides incomplexes with nucleic acid encoding them such as in the context ofphage display.

BACKGROUND TO THE INVENTION

The generation of molecules with high affinity and specificity forbiological targets is a central problem in chemistry, biology andpharmaceutical sciences. In particular, binding ligands are importantfor the creation of drugs that can intervene with biological processes.The creation of ligands that bind to a chosen target ligand usuallyinvolves a process of generating a plurality of putative bindingmolecules and testing said molecules for their binding properties.

While biological in vitro selection techniques were used efficiently forthe isolation of large biopolymeric structures such as antibodies, theywere less practicable for the isolation of small molecule drugs to date.Biological in vitro selection techniques are generally limited tobiological polymers such as polypeptides, RNA or DNA. Short biopolymersas for example peptides can also bind to biological targets but they cansuffer from conformational flexibility and may be prone to proteolyticdegradation in bodily fluids. In addition, binding affinities of shortlinear peptides are often weak. Various circularization strategies areknown to constrain genetically encoded small peptide libraries. Phagedisplayed peptide repertoires are known for example to be circularizedby the oxidation of two flanking cysteine residues. mRNA encoded cyclicpeptide libraries are known to be generated by linking the N-terminalamine and a lysine residue of the peptide with a chemical cross-linkingreagent. This strategy was used for the isolation of redox-insensitivemacrocycles that bind to the signaling protein Gαil (Millward, S. W. etal., ACS Chem. Biol., 2007). Various strategies are also known for useto incorporate non-natural building blocks into genetically encodedpolypeptide libraries to expand the diversity of the libraries or toinsert properties that can not be provided by natural amino acids.However, the strategies allowed only the addition of a limited number ofsmall organic appendages to linear genetically encoded polypeptides.Frankel, A. et al., for example had incorporated non-natural amino acidsinto natural polypeptides that were encoded by mRNA display (Frankel, A.et al., Chem. Biol., 2003). Jespers L. et al. had chemically linked afluorescent reporter molecule to a hypervariable loop of an antibodyrepertoire displayed on phage, and selected this repertoire for antigenbinding (Jespers, L., et al., Prot. Eng., 2004). Dwyer, M. A. et al. hadjoined synthetic peptides to a repertoire of phage displayed peptides bynative chemical ligation for the generation of a protease inhibitorlibrary containing a non-natural amino acid (Dwyer, M. A. et al.,Chemistry & Biology, 2000). Small organic molecules have also beenlinked to mRNA encoded combinatorial peptide repertoires. The researchteam of Roberts, R. W. had attached a penicillin moiety to a fixedposition of an mRNA-display peptide library to select inhibitors of theStaphylococcus aureus penicillin binding protein 2a (Li, S. and Roberts,W. R., Chem. & Biol., 2003).

In order to apply in vitro selection to combinatorial compound librarieshaving more diverse molecule architectures (e.g. branched molecules) andbeing formed of non-natural building blocks, various methodologies havebeen proposed. Unlike biological in vitro selection methods, thesemethodologies use chemical strategies to attach DNA tags to smallorganic molecules. Brenner S. and Lerner R. A. had proposed a process ofparallel combinatorial synthesis to encode individual members of a largelibrary of chemicals with unique nucleotide sequences on beads (Brenner,S. and Lerner, R. A., PNAS, 1992). After the chemical entity is bound tothe target, the genetic code is decoded by sequencing of the nucleotidetag. Liu D. R. and co-workers had conjugated a small collection oforganic molecules to DNA oligonucleotides and performed affinityselections with different antigens (Doyon, J. B. et al., JACS, 2003).Neri D. and co-workers had generated large repertoires of molecule pairsby self-assembly of smaller DNA encoded chemical sub-libraries throughhybridization of two DNA strands (Melkko, S. et al., Nature Biotechnol.,2004). The methodology was successfully used for affinity maturation ofsmall molecule ligands. Halpin D. R. and Harris P. B. developed astrategy for the in vitro evolution of combinatorial chemical librariesthat involves amplification of selected compounds to perform multipleselection rounds (Halpin, D. R. and Harbury, P. B., PLOS Biology, 2004).Woiwode T. F. et al. attached libraries of synthetic compounds to coatproteins of bacteriophage particles such that the identity of thechemical structure is specified in the genome of the phage (Woiwode, T.F., Chem. & Biol., 2003). All these strategies employing DNA specifiedchemical compounds have proven to be efficient in model experiments andsome have even yielded novel small molecule binders. However, it becameapparent that the encoding of large compound libraries and theamplification of selected compounds is much more demanding than theequivalent procedures in biological selection systems.

Jespers et al (2004 Protein engineering design and selection, volume 17,no. 10, pages 709-713) describes the selection of optical biosensorsfrom chemisynthetic antibody libraries. This document is concerned withthe attachment of a fluorescent reporter molecule through thehypervariable loop of an antibody repertoire displayed on the phage. Inparticular, this document describes linking of a fluorescent reportermolecule into a hypervariable loop (complementarity determining regionor CDR) of a synthetic antibody repertoire. The fluorescent reportermolecule is linked by a single covalent bond to an artificiallyintroduced cysteine residue in the hypervariable loop. A one to oneattachment is performed. The cysteine residues on the phage particleswere reduced with DTT and the excess reducing agent was removed byconventional polyethylene glycol (PEG) precipitation as is well known inthe art.

Dwyer et al disclose biosynthetic phage display, describing a novelprotein engineering tool combining chemical and genetic diversity. Dwyeret al (Chem Biol 2000, volume 7, no. 4, pages 263-274) describe thechemical ligation of a synthetic peptide having a non-natural amino acidonto a library of synthetic peptides comprising the main structuralresidues of a protein of interest. The motivation for performing thiswas in order to generate a diverse range of protease sequences, eachhaving a constant segment incorporating an unnatural amino acid. Thesynthetic peptide comprising the non-natural amino acid was simplyjoined by native chemical ligation, resulting in coupling of the twopeptide fragments together. No connector compound is disclosed. No smallmolecule attachment is disclosed. No constraint or conformationalrestriction of the resulting polypeptide was achieved. No covalentbonding of particular moieties to the polypeptide chain is disclosed.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp, D. S. andMcNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., ChemBioChem,2005). Meloen and co-workers had used tris(bromomethyl)benzene andrelated molecules for rapid and quantitative cyclisation of multiplepeptide loops onto synthetic scaffolds for structural mimicry of proteinsurfaces (Timmerman, P. et al., ChemBioChem, 2005). Methods for thegeneration of candidate drug compounds wherein said compounds aregenerated by linking cysteine containing polypeptides to a molecularscaffold as for example tris(bromomethyl)benzene are disclosed in WO2004/077062 and WO 2006/078161.

Methods provided in WO 2004/077062 and WO 2006/078161, are based onsampling individual compounds for example in a screening procedure.Screening of individual compounds or small sets of compounds is tediousand can be expensive if large numbers of compounds are analyzed. Thenumber of compounds that can be assayed with screening assays generallydoes not exceed several thousands. Moreover, reaction conditionsdescribed in WO 2004/077062 to tether a cysteine containing peptide to ahalomethyl containing scaffold as for example tris(bromomethyl)benzeneare not suitable to modify a genetically encoded cysteine containingpeptide.

WO2004/077062 discloses a method of selecting a candidate drug compound.In particular, this document discloses various scaffold moleculescomprising first and second reactive groups, and contacting saidscaffold with a further molecule to form at least two linkages betweenthe scaffold and the further molecule in a coupling reaction. Thismethod suffers from many restrictions. Firstly, it is based on the useof synthetic peptides and in vitro chemical reactions in separatevessels. For this reason, it is labour intensive. There is noopportunity to automate or to apply the method to the screening of manypeptide variants without manually producing each variant by conductingnumerous parallel independent reactions. There is no mention ofgenetically encoded diversity in this document, and certainly no mentionof application to genetically encoded phage libraries. Indeed, thereaction conditions disclosed in this document mean that it would bedifficult or impossible to perform the reactions disclosed on phageparticles.

WO2006/078161 discloses binding compounds, immunogenic compounds andpeptidomimetics. This document discloses the artificial synthesis ofvarious collections of peptides taken from existing proteins. Thesepeptides are then combined with a constant synthetic peptide having someamino acid changes introduced in order to produce combinatoriallibraries. By introducing this diversity via the chemical linkage toseparate peptides featuring various amino acid changes, an increasedopportunity to find the desired binding activity is provided. FIG. 7 ofthis document shows a schematic representation of the synthesis ofvarious loop peptide constructs. There is no disclosure of geneticallyencoded peptide libraries in this document. There is no disclosure ofthe use of phage display techniques in this document. This documentdiscloses a process which is considered to be incompatible with phagedisplay. For example, the chemistry set out in this document is likelyto result in the linking molecule reacting with the phage coat. There isa risk that it could cross link phage particles. It is probable thatphage particles would be inactivated (e.g. would lose their infectivity)if subjected to the chemistry described. This document is focussed onthe manipulation of various synthetic peptides in independent chemicalconjugation reactions.

Millward et al (2007 Chemical Biology, volume 2, no. 9, pages 625-634)disclose the design of cyclic peptides that bind protein surfaces withantibody like affinity. This document discloses cyclisation of variouspeptides produced from a genetically encoded library. The polypeptidesare cyclised through reaction of a chemical cross-linker with theN-terminal amine and an amine of a lysine in the polypeptide. In thisdocument, the genetically encoded library is a mRNA display library.This document does not disclose the attachment of any connector compoundto the resulting polypeptides. This document is concerned with theproduction of redox insensitive cyclised peptides. The chemistrydisclosed in this document is cyclisation through reaction of a chemicalcross linker with the N-terminal amine and an amine of a lysine providedin the polypeptide. The cyclisation reaction is performed in a 50milimolar phosphate buffer at pH 8 by the addition of DSG (1 mg per mlin DMF). At most, this document discloses the bridging of two parts of apolypeptide chain via a cross linking moiety in order to provide acyclic peptide.

US2003/0235852 discloses nucleic acid-peptide display librariescontaining peptides with unnatural amino acid residues, and methods ofmaking these using peptide modifying agents. In other words, thisdocument discloses genetically encoded polypeptide libraries thatcontain either a non-natural amino acid or an amino acid where anon-natural building block (e.g. penicillin) is post-translationallyattached in a chemical reaction. This document is focused on knownmethods for associating a translated peptide with the nucleic acid whichencoded it. The further problem addressed by this document is how toincorporate unnatural amino acids into that peptide. This is principallyaccomplished by the use of suppressor tRNAs in order to incorporateunnatural amino acids in response to amber/ochre/opal codons as is wellknown in the art. In other more minor embodiments, unnatural amino acidsare created post-translationally by treatment of the translated peptidewith a ‘peptide modifying agent’. This reagent is typically aimed ataltering an existing amino acid residue in order to convert it into anunnatural amino acid residue, or otherwise render it functionallyreactive or receptive to the attachment of a further chemical moiety.Specifically, this document teaches the post-translational conjugationof a cysteine residue in the polypeptide of interest to the beta lactamantibiotic 6-bromoacetyl penicilamic acid. This results in theconjugation of this penicillin analogue onto the polypeptide of interestvia a single bond to the cysteine residue side chain. No multiplebonding of the molecule being ligated to the polypeptide is disclosed.No conformational constraint of the polypeptide is described. No peptideloops or any other complex tertiary structures are formed by the methodsdisclosed in this document—it is purely a way of attaching a singlefurther molecular group to a polypeptide via a single bond. Conventionalconjugation chemistry is used in order to perform the modifications tothe polypeptides in this document.

SUMMARY OF THE INVENTION

The present invention advantageously allows the combination ofgenetically encoded diversity, in particular genetically encodedpolypeptide libraries, with chemical modification and conformationalconstraint.

Moreover, the techniques disclosed herein provide for the first time thelinking of a connector compound to a polypeptide molecule by at leastthree covalent bonds. This provides the advantage of conformationalconstraint of the polypeptide, in particular conformational constraintof at least two segments of the polypeptide with respect to each other.By contrast, cross-linking techniques of the prior art, or the use of aconnector which makes only two covalent bonds, will constrain only asingle segment of the polypeptide.

Advantages of the invention flow from these technical features, forexample due to their triple-bonded construction, the conjugatedmolecules of the invention have two or more peptide loops that caninteract with a target. With multiple binding loops, higher bindingaffinities can be obtained than with molecules that have just a singlepeptide loop.

In addition, the interaction surface of a molecule of the invention withtwo or more binding loops for interaction with a target is larger thanthe one of a molecule with a single peptide loop with a target. Thelarger binding surface can provide improved binding affinity, and/or canalso provide improved specificity.

Thus in one aspect the invention provides a complex comprising

-   -   (i) a polypeptide;    -   (ii) a nucleic acid encoding the polypeptide of (i);    -   (iii) a connector compound attached to said polypeptide    -   wherein said connector compound is attached to the polypeptide        by at least three discrete covalent bonds.

More in particular the invention provides a complex comprising a phageparticle, said phage particle comprising

-   -   (i) a polypeptide;    -   (ii) a nucleic acid encoding the polypeptide of (i);    -   (iii) a connector compound attached to said polypeptide        wherein said connector compound is attached to the polypeptide        by at least three discrete covalent bonds.

The covalent bonds are suitably discrete covalent bonds in the sense ofeach being a separate bond between the connector compound and a part ofthe polypeptide. For example, a single bridge between the polypeptideand the connector compound which single bridge is made up of threecovalent bonds (e.g. connector -x-y-polypeptide where “-” represents acovalent bond) would not be considered to comprise at least threediscrete covalent bonds because the three bonds are not three separatebridges or connections from the connector compound to the targetpolypeptide. The underlying principle is that the connectorcompound/molecular core and the polypeptide are joined by at least threeseparate covalent bridging bonds.

Suitably each of the at least three covalent bonds is formed with aseparate amino acid residue of the polypeptide. In other words, aseparate amino acid residue is suitably an individual or distinct aminoacid residue—more than one bond may be formed with a single species ortype of amino acid residue e.g. two of the bonds may each be formed withcysteine residues but suitably those two cysteine residues will beseparate cysteine residues.

The connector compound-polypeptide part of the complex described aboveis sometimes referred to as the ‘conjugate’. In some embodiments theconjugate (i.e. a polypeptide-connector compound moiety corresponding tothat comprised by the complex of the invention) may be separatelysynthesised. In this embodiment the conjugate may not be complexed witha nucleic acid. This is discussed in more detail below.

Suitably ‘encoding’ has its natural meaning in the art, i.e. encoding inthe sense of the universal triplet code to convert nucleotide sequenceinto polypeptide sequence. In the prior art, ‘encoding’ might have beenused in the sense of ‘tagging’ or ‘deconvoluting’ e.g. when a uniquenucleotide sequence is used to tag a moiety and that knowledge of thenucleotide sequence can ‘decode’ i.e. tell the user which tagged moietywas present, yet without bearing any biological relationship to itsstructure. However, in the present invention, ‘encode’ and ‘decode’ areused in the traditional natural manner to refer to encoding in the senseof translation from nucleotide sequence to amino acid sequence.

Suitably the connector compound comprises an organic molecule. Suitablythe connector compound comprises a small organic molecule.

Suitably the covalent bonds are formed between the connector compoundand amino acid residues of the polypeptide.

Suitably said polypeptide comprises a cysteine residue, and suitably atleast one of said three discrete covalent bonds for attachment of saidconnector compound to the polypeptide comprises a bond to said cysteineresidue.

Suitably the connector compound has molecular symmetry corresponding tothe number of covalent bonds by which it is attached to the polypeptide.

Suitably the connector compound possesses threefold molecular symmetryand the connector compound is attached to the polypeptide by threecovalent bonds.

Suitably the connector compound comprises a structurally rigid chemicalgroup.

Suitably the connector compound comprises tris-(bromomethyl)benzene(TBMB).

In another aspect, the invention relates to a complex as describedabove.

Suitably said polypeptide is an mRNA displayed polypeptide.

Suitably said polypeptide is comprised by a phage particle.

Nucleic acid has its usual meaning in the art and may comprise DNA, RNAor any other suitable nucleic acid. Nucleic acid may compriseoligonucleotides(s) or phage genome(s) or any other suitable example ofnucleic acids known to the skilled worker.

Suitably said nucleic acid is comprised by said phage particle.

In another aspect, the invention relates to a genetically encodedpolypeptide library comprising at least two different complexes asdescribed above.

In another aspect, the invention relates to a method for making acomplex, said method comprising

(i) providing a polypeptide

(ii) providing a connector compound

(iii) attaching said connector compound to said polypeptide by formationof at least three covalent bonds between said connector compound andpolypeptide.

Suitably the reactive groups of said polypeptide are reduced, andsuitably the polypeptide comprising reduced reactive groups is purifiedby filtration before step (iii).

Suitably when the reactive groups comprise cysteine they are reduced; inthis embodiment the purification is purification from reducing agent,for example by filtration.

Suitably following the filtration purification step, the polypeptide ismaintained in the reduced state for bonding to the connector compound byincubation in degassed buffer and in the presence of chelating agent.

Suitably step (iii) comprises incubation of the polypeptide andconnector compound together at 30° C. at pH 8 in aqueous buffercomprising acetonitrile.

Suitably the polypeptide is comprised by a phage particle.

Suitably the connector compound comprises tris-(bromomethly)benzene(TBMB).

Suitably the tris-(bromomethly)benzene is present at 10 μm.

Suitably the tris-(bromomethly)benzene is present at 10 μm, thechelating agent is ethylenediaminetetraaceticacid (EDTA), theacetonitrile is present at 20% and the incubation step (iii) isconducted for 1 hour.

Suitably said method comprises the further step of (iv) cleaving one ormore bonds of the polypeptide chain. This has the advantage of modifyingthe polypeptide chain. For example, this may have the benefit ofproducing multiple polypeptides attached to a single connector compounde.g. when the cleavage takes place on the polypeptide chain in betweenbonds between the polypeptide and the connector compound.

Suitably said cleavage step comprises contacting said polypeptide with aprotease.

In another aspect, the invention relates to a complex obtained by amethod as described above.

In another aspect, the invention relates to a method for identifying acomplex according to any preceding claim which is capable of binding toa ligand, the method comprising

(i) providing a complex as described above

(ii) contacting said complex with the ligand, and

(iii) selecting those complexes which bind said ligand.

Such selection method may be conducted in any suitable format. Suitablythe ligand is immobilised. The complex is then contacted with theimmobilised ligand. Non-binding complex(es) are then washed away. Inthis manner, those complexes which bind the immobilised ligand areenriched or selected. In one embodiment it is possible that thecomplexes may be recovered by release of the ligand i.e. releasing oreluting the complex-ligand moiety. However, suitably the complexes arerecovered by elution (separation) from the immobilised ligand. In thisembodiment the eluted complexes are no longer bound to the ligand at theelution step.

The complexes, or the polypeptide(s) of said complexes, or thepolypeptide-connector compound conjugates of said complexes, may beuseful in other settings. For example they may be useful as a basis forthe design of drugs such as small drugs, or may be useful as CDRs or asbinding moieties (e.g. for tagging or detection of their bindingpartner(s)) or other applications where the intimate knowledge of theinteraction can be exploited.

In another aspect, the invention relates to a method as described abovefurther comprising determining the sequence of the nucleic acid of saidcomplex.

In another aspect, the invention relates to a method as described abovefurther comprising the step of manufacturing a quantity of the complexisolated as capable of binding to said ligand.

In another aspect, the invention relates to a method as described abovefurther comprising the step of manufacturing a quantity of thepolypeptide-connector compound moiety comprised by the complex isolatedas capable of binding to said ligand. In this embodiment thepolypeptide-connector compound moiety may be advantageously synthesisedin the absence of nucleic acid.

In another aspect, the invention relates to a method as described abovefurther comprising the step of manufacturing a quantity of a polypeptideisolated or identified by a method of the invention, said manufacturecomprising attaching the connector compound to the polypeptide, whereinsaid polypeptide is recombinantly expressed or chemically synthesized.In another embodiment the invention relates to a method as describedabove further comprising the step of manufacturing a quantity of apolypeptide isolated or identified by a method of the invention, saidmanufacture comprising attaching a connector compound to thepolypeptide, wherein the connector compound may be different from theconnector compound attached during isolation or identification of thepolypeptide, provided that said connector compound is attached to saidpolypeptide by at least three covalent bonds, and wherein saidpolypeptide is recombinantly expressed or chemically synthesized.

In another aspect, the invention relates to a conjugate comprising

(i) at least two polypeptide molecules, and

(ii) at least one connector compound molecule,

wherein said at least two polypeptide molecules are each attached tosaid connector compound molecule by at least one covalent bond. Suitablysaid connector compound is bonded to said at least two polypeptidemolecules by a total of at least three discrete covalent bonds.

In another aspect, the invention relates to a conjugate as describedabove wherein said conjugate comprises at least three polypeptidemolecules, and wherein said at least three polypeptide molecules areeach attached to said connector compound molecule by at least onecovalent bond. Suitably said connector compound comprisestris-(bromomethly)benzene (TBMB).

In another aspect, the invention relates to a human plasma kallikreininhibitor comprising an amino acid sequence selected from the groupconsisting of ACSDRFRNCPLWSGTCG (SEQ ID No. 1), ACSTERRYCPIEIFPCG (SEQID No. 2), ACAPWRTACYEDLMWCG (SEQ ID No. 3), ACGTGEGRCRVNWTPCG (SEQ IDNo.4), and ACSDRFRNCPADEALCG (SEQ ID No. 5).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows generation of phage encoded combinatorial small chemicallibraries. A phage encoded peptide with three cysteine residues istethered to the tri-functional compound tris-(bromomethyl)benzene in anucleophilic substitution reaction. The resulting chemical entities maybe further modified through enzymatic reactions.

FIGS. 2A and 2B show assessment of the reaction conditions for linkingphage displayed peptides to tris-(bromomethyl)benzene (TBMB). FIG. 2A:Molecular mass of the GCGSGCGSGCG (SEQ ID No. 15)-D1-D2 fusion proteinbefore and after reaction with 10 μM TBMB in 20 mM NH₄HCO₃, 5 mM EDTA,pH 8, 20% ACN at 30° C. for 1 hour determined by mass spectrometry. Themass difference of the reacted and non-reacted peptide fusion proteincorresponds to the mass of the small molecule core mesitylene. FIG. 2B:Titres (transducing units) of phage reduced and treated with variousconcentrations of TBMB in 20 mM NH₄HCO₃, 5 mM EDTA, pH 8, 20% ACN at 30°C. for 1 hour. Titres of phage from fdg3p0ss21 (black) and from library1 (white) are shown.

FIGS. 3A, 3B and 3C show phage library design and sequences of selectedclones. FIG. 3A: Amino acid sequence of peptide fusion proteinsexpressed by clones of library 1. The leader sequence is removed uponsecretion of the protein by an E. coli protease leaving a peptide withan N-terminal alanine, two random 6-amino acid sequences flanked bythree cysteines and a Gly-Gly-Ser-Gly (SEQ ID No. 20) linker thatconnects the peptide to the gene-3-protein. FIGS. 3B and 3C: Amino acidsequences of clones selected with human plasma kallikrein (B) andcathepsin G (C). Inhibitory activities of the TBMB modifiedpeptide-D1-D2 fusion proteins are indicated. Sequence similarities arehighlighted.

FIGS. 4A and 4B show affinity maturation of human plasma kallikreininhibitors. FIG. 4A: Design of library 2, 3 and 4. In each library, oneof the peptide loops has the sequence of a consensus motif identified inthe first selections and the other contains six random amino acids. FIG.4B: Amino acid sequences of clones selected with human plasmakallikrein. All clones derive from library 2. The inhibitory activitiesof TBMB modified peptide-D1-D2 fusion proteins are indicated. The boxedareas highlight sequence similarities in the second binding loop.

FIG. 5 shows inhibition of human plasma kallikrein by TBMB modifiedsynthetic peptides. The inhibitory activity is expressed as thefractional activity (inhibited rate/uninhibited rate) at varyinginhibitor concentrations.

FIG. 6 shows representative NMR solution structure of TBMB modifiedpeptide PK15 shown as a ‘sausage’ structure. The peptide loops 1 and 2are shown. The alpha carbon atoms of the amino acids in the peptideloops and at the termini are shown as spheres.

FIGS. 7A, 7B, 7C and 7D show chemical reactions of the tri-functionalcompound TBMB with peptides containing one or two cysteines. FIG. 7A:Plausible reaction mechanism of TBMB with a peptide fusion proteincontaining two cysteine residues. FIG. 7B: Mass spectra of a peptidefusion proteins with two cysteines before and after reaction with TBMB.FIG. 7C: Plausible reaction mechanism of TBMB with a peptide fusionprotein containing one cysteine residue. FIG. 7D: Mass spectra of apeptide fusion proteins with one cysteine before and after reaction withTBMB.

FIGS. 8A, 8B and 8C show inhibition of contact activation in humanplasma by aprotinin and TBMB modified peptide PK15. Effect of aprotinin(FIG. 8A) and TBMB modified peptide PK15 (FIG. 8B) on thrombingeneration triggered by actin. Both inhibitors cause dose-dependentprolongation of lag time compared to the control sample. FIG. 8C: Thesum of the activities of factor XIIa and plasma kallikrein was measuredwith the colorimetric substrate H-D-Pro-Phe-Arg-pNA in human plasma ofthree different donors treated with varying concentrations of inhibitor.Contact activation was initiated by addition of kaolin. Mean values andstandard deviations are indicated.

FIG. 9a shows generation of phage encoded combinatorial small chemicallibraries. A phage encoded peptide with three cysteine residues istethered to the tri-functional compound tris-(bromomethyl)benzene in anucleophilic substitution reaction. The resulting chemical entitiescould optionally be further modified through enzymatic reactions. FIG.9b shows the chemical structure of a macrocyclic plasma kallikreininhibitor isolated by phage display (PK15).

FIGS. 10a and 10b show assessment of the reaction conditions for linkingphage displayed peptides to tris-(bromomethyl)benzene (TBMB). FIG. 10a :Molecular mass of the GCGSGCGSGCG (SEQ ID No. 15)-D1-D2 fusion proteinbefore and after reaction with 10 μM TBMB in 20 mM NH₄HCO₃, 5 mM EDTA,pH 8, 20% ACN at 30° C. for 1 hour determined by mass spectrometry. Themass difference of the reacted and non-reacted peptide fusion proteincorresponds to the mass of the small molecule core mesitylene. FIG. 10b: Titres (transducing units) of phage that were reduced and treated withvarious concentrations of TBMB in 20 mM NH₄HCO₃, 5 mM EDTA, pH 8, 20%ACN at 30° C. for 1 hour. Titres of phage from fdg3p0ss21 (black) andfrom library 1 (white) are shown.

FIGS. 11a, 11b and 11c show phage library design and sequences ofselected clones. FIG. 11a : Amino acid sequence of peptide fusionproteins expressed by clones of library 1. The leader sequence isremoved upon secretion of the protein by an E. coli protease leaving apeptide with an N-terminal alanine, two random 6-amino acid sequencesflanked by three cysteines and a Gly-Gly-Ser-Gly (SEQ ID No. 20) linkerthat connects the peptide to the gene-3-protein. FIGS. 11b and 11c :Amino acid sequences of clones selected with human plasma kallikrein (b)and cathepsin G (c). Inhibitory activities of the TBMB modifiedpeptide-D1-D2 fusion proteins are indicated. Sequence similarities arehighlighted.

FIGS. 12a and 12b show affinity maturation of human plasma kallikreininhibitors. FIG. 12a : Design of library 2, 3 and 4. In each library,one of the peptide loops has the sequence of a consensus motifidentified in the first selections and the other contains six randomamino acids. FIG. 12b : Amino acid sequences of clones selected withhuman plasma kallikrein. All clones derive from library 2. Theinhibitory activities of TBMB modified peptide-D1-D2 fusion proteins areindicated. The sequence similarities in the second binding loop arehighlighted.

FIG. 13 shows inhibition of human plasma kallikrein by TBMB modifiedsynthetic peptides. The inhibitory activity is expressed as thefractional activity (inhibited rate/uninhibited rate) at varyinginhibitor concentrations. Clones PK2, PK4, PK6 and PK13 were isolated inphage selections using library 1. PK15 derives from library 2 and is anaffinity matured inhibitor.

FIG. 14 shows NMR solution structure of TBMB modified peptide PK15. Thepeptide loops 1 and 2 are shown. The mesitylene core, the three cysteineresidues and the terminal alanine (N-terminus) and glycine (C-terminus)are shown in the center of the structure in the area marked with dots.The backbone atoms of the peptide are represented as a sausage and theside chains of the amino acids are shown as sticks.

FIGS. 15A, 15B, 15C and 15D show chemical reactions of thetri-functional compound TBMB with peptides containing one or twocysteine residues. FIG. 15A: Plausible reaction mechanism of TBMB with apeptide fusion protein containing two cysteine residues. FIG. 15B: Massspectra of a peptide fusion protein with two cysteines before and afterreaction with TBMB. FIG. 15C: Plausible reaction mechanism of TBMB witha peptide fusion protein containing one cysteine and one lysine residue.FIG. 15D: Mass spectra of a peptide fusion protein with one cysteine andone lysine residue before and after reaction with TBMB.

FIG. 16 shows suppression of factor XII activation in human plasmathrough the inhibition of plasma kallikrein with aprotinin or TBMBmodified peptide PK15. The intrinsic coagulation pathway in human plasmaof three different donors was initiated by addition of kaolin. Thenegatively charged surface of kaolin activates small amounts of factorXII. Prekallikrein is converted to kallikrein by activated factor XII(XIIa), and kallikrein exerts a positive feedback to activate morefactor XII. The activity of factor XIIa was measured with thecolorimetric substrate H-D-Pro-Phe-Arg (SEQ ID No. 37)-pNA. Mean valuesand standard deviations of factor XIIa activity are indicated.

DETAILED DESCRIPTION OF THE INVENTION

The invention brings novel features and attendant advantages which canbe explained in more detail in connection with the generation ofgenetically encoded molecules with a core structure. In particular, theinvention provides conformational restraint which is not achieved byknown peptide cyclisation techniques. Moreover, the cross-linker inknown systems such as those of Roberts (ibid) does not have thecharacter of a central core/connector compound of the present invention.In the known systems, the cross-linker was used purely to replace adisulfide bond to generate a redox-insensitive cyclic peptide. There isno mention or suggestion of the concept of a central core with multipleappendages such as a triple covalently bonded connectorcompound-polypeptide complex as is taught herein. Indeed, it must benoted that in the present invention the polypeptide is linked to thecore structure via at least three covalent bonds, providing a keystructural difference compared to known systems. The linkage of a corestructure (connector compound) to a genetically encoded polypeptide viathree or more bonds is a complex reaction that has not been shownbefore.

Furthermore, the linkage of a polypeptide to a connector compound via atleast three covalent bonds could yield several different products. Thiscould cause difficulties in the selection process and in the decodingprocedure. However, according to the present invention, a solution isprovided using a connector compound with three reactive groups andpreferably 3-fold rotational symmetry, which combination has theadvantage of yielding a single product. Of course the skilled readerwill appreciate that in certain obscure circumstances a connectorcompound with a 3-fold rotational symmetry may yield multiple products,most notably in the example of a tetrahedral molecule with threeidentical reactive groups; this also has a 3-fold rotational symmetrybut it would yield two stereoisomers. Nevertheless, for ease ofunderstanding such theoretical exceptions to the formation of a singleproduct are acknowledged to be possible, suitably connector compoundswith a 3-fold rotational symmetry yield a single product according tothe present invention; in the rare circumstances noted above thensuitably the polypeptide is chosen to avoid tetrahedral moleculeformation and therefore maintain formation of only a single product.

The connector compound used in methods and compositions described hereinis different from known bivalent cross-linkers (e.g. as used by Millwardet al. ibid.) in the key requirement that a connector compound of theinvention has at least three reactive groups that can form at leastthree covalent bonds with the target polypeptide. This feature yieldsnumerous technical benefits to the invention. Firstly, by bonding theconnector compound to the polypeptide via at least three covalent bonds,at least two polypeptide loops are created. These loops are formedbetween the first and second bonds, and between the second and thirdbonds of the connector compound to the polypeptide. The known linkerdescribed by Millward et al. can only connect two functional groups of apeptide, and cannot form two or more constrained peptide loops.

Advantages of Connector Compound—Polypeptide Bonding

There are a number of properties that distinguish molecules of theinvention having 3 or more linkages to a connector compound from othermolecules such as those with only 2 linkages. Some of these areexplained below.

Firstly, it will be appreciated that molecules with two linkages to aconnector compound are constrained through linking the flexible ends ofa linear peptide together. This is also the case for molecules accordingto the present invention with 3 or more covalent bonds to a connectorcompound. However, the conformation of molecules according to thepresent invention with 3 or more covalent bonds to a connector compoundis constrained by two additional effects that do not apply to a moleculewith just two links:

i) the polypeptide bonded to the connector compound via at least threecovalent bonds will comprise at least two constrained polypeptide loops

ii) the polypeptide loops can interact with each other throughnon-covalent interactions to generate additional constraint, and

iii) each of the loops occupies space that can not be occupied by theother loop(s) which additionally restricts their conformationalflexibility.

In order to illustrate these points, the possible paths that can betaken by a polypeptide anchored at points A and C to a connectorcompound can be imagined. The introduction of an anchor point B, betweenpoints A and C, and to the same connector compound, will further limitthe possible paths taken by the polypeptide, and thereby itsconformational entropy. As binding of the peptide to a ligand requiresloss of conformational entropy (and provided the peptide can adopt aconformation that is complementary to a ligand), the binding affinitybetween the peptide ABC constrained at the intermediate point B and theligand is expected to be higher than the peptide AC. Thus, higherbinding affinities are achievable using the constrained molecules of thepresent invention than has been possible in the prior art.

In addition to these key points, further advantages of the three or morecovalent linkages between polypeptide and connector compound are set outbelow.

The molecules of the invention can bind to a target through theinteraction of two or more conformationally constrained peptide loops.The more binding loops, the higher affinities and specificities can beobtained. A parallel effect occurs with antibodies—they bind best whenmultiple CDRs interact with the target. The molecules according to thepresent invention thus advantageously provide this technical benefit ofmultiple loops for interaction, which benefit is absent from moleculeswith fewer than three bonds.

In addition to the actual provision of a second (or subsequent) peptideloop, it is important to note that such a loop also brings the advantageof conformationally constraining the other loop(s). This can be throughoccupying some of the limited three-dimensional space which can then notbe occupied by the other loop(s). Alternatively this can be throughnon-covalent interactions between the multiple loops.

From these advantages it can also be noted that more structured ligandsgenerally bind with higher affinities (less entropy is lost uponbinding) and specificities.

As discussed herein, the invention also provides for the production oflooped peptide structures in which each of the two (or more) loops has adifferent property. Such structures are referred to as “dual specifics”to reflect the fact that a single molecular entity has dualspecificities attributable to two different parts (loops) of the sameoverall structure. The advantage of such embodiments is that each loopcan be selected or constructed to bind to a different target (such as an“antigen”). This represents a further distinguishing feature of a threebond system according to the present invention.

In addition to these effects, the molecules of the invention alsoprovide the possibility of sandwiching a single antigen (or otherentity) between two segments of polypeptide chain. This possibility isof course absent from polypeptide constructs with fewer than two loops.Of course the particular arrangement adopted may depend on the geometryof the particular construct being used, but the invention renders thispossible which is in contrast to prior art techniques.

Of course the above discussion has made mention of the loops generatedaccording to the present invention. In some embodiments, those loops arethen cleaved. It is important to note that even in such embodiments, theloops are formed, it is simply that the looped molecule is treated as anintermediate which is then further processed by cleavage of the loops toproduce a tethered-multiple-linear-peptide structure. In theseembodiments, because the peptide is initially linked to the connectorcompound via three or more covalent bonds, after peptide cleavage themolecules will be decorated with three or more peptide moieties. Suchmolecules can form more interactions to targets and higher bindingaffinities/specificities are expected, which is a further advantage ofthe three-bonded system of the invention.

It is an advantage that molecules of the invention having two or morepolypeptide loops can form more interactions with a target ligand andtherefore can have higher affinities and/or specificities thanpolypeptide molecules with only a single loop. For example, it may bedesirable to refine the second loop for better affinity, which isclearly not possible for single-loop molecules.

It is an advantage that in the complexes of the invention, the connectorcompound holds at least two polypeptide loops in close spatialproximity. These two or more loops can interact simultaneously withdifferent epitopes on the same target ligand.

Moreover, the benefit of having two or more loops can be exploited inthe manufacture of ‘dual specific’ molecules, where one loop has or isselected for a particular property or binding affinity, and the otherloop for a different property or affinity. These molecules are referredto as “bispecifics” or “dual specifics”. There are several typespossible. For example,

(a) bispecifics made by selecting on loop 1 and then on loop 2 (or more)

(b) two linked bicyclic macrocycles

(c) one bicyclic macrocycle plus peptide or drug.

For (a), this might typically be done by making/selecting one aliquot ofa library against a first antigen, and another aliquot against a secondantigen. The selected loops could then be combined pairwise, for exampleby standard techniques such as recombining the nucleic acid segmentsencoding the two loops to provide a new library of differentcombinations of first and second loops. The pairwise combined molecules(e.g. phage) may then be screened and/or selected for binding againstboth antigens sequentially. In this way, bispecifics capable of bindingto two separate antigens may be made. Naturally this method can beaugmented with further optional steps such as the binding affinities foreach antigen could be improved by mutation of each loop, which may bedirected or even random mutation.

In a variation of this technique, one aliquot of library could beselected for binding to a first antigen. The loop most important forbinding could be identified, for example by inspection of “consensussequences” among those selected as binders, and the other loop could berandomized and selected against the second antigen.

Most suitably bispecifics of this type such as described in (a) would bemade on phage.

Variant molecules noted in (b) and (c) above could equally be made asphage (in a similar manner to above). Alternatively, most likely the twolinked entities could be selected separately then fused at the step ofchemical synthesis, which might simplify their selection/construction.

It is a further significant advantageous feature of aspects of theinvention that, in addition to the connector compound of the inventionserving to connect polypeptide segments through covalent bonds via theamino acid residues at the base of each peptide loop, the connectorcompound also engages in further non-covalent interactions (such asionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waalsinteractions) with additional elements of the polypeptide chain such asother amino acid residues. By contrast, the bivalent linker of Millwardet al. is linear and highly flexible (propyl), and its sole role is toconnect two ends of a polypeptide to create a redox-insensitive cyclicpeptide. The linker of Millward et al. would not be expected to makesignificant non-covalent interactions with the polypeptide loop, as itis small and highly flexible, and indeed there is no evidence that itdoes. This advantage of the invention is further illustrated in theexamples section, together with evidence of the advantageous noncovalentinteractions. Thus suitably the polypeptide is joined to the connectorcompound by one or more non-covalent interactions, in addition to thecovalent bond(s) discussed herein. This has the further advantage ofproviding additional level of structural constraint to thecomplex/conjugate of the invention.

It is an advantage of the invention that a molecule with multiplepeptide loops is generally more structured than a polypeptide with asingle peptide loop. Highly structured molecules tend to be morespecific. Also, well structured molecules have generally better bindingaffinities. In addition, a molecule with multiple peptide loops can formmore interactions with a target ligand than a polypeptide with a singlepeptide loop.

It is a further benefit of aspects of the invention that the connectorcompound of the invention also imposes conformational constraintsderiving from its own chemical structure. For example, some chemicalgroups are known to be inflexible, to prevent rotation, to providesteric hindrance or restriction, to present a rigid structure orotherwise to provide scaffold or constraint to the complex. Thussuitably the connector compound of the invention comprises a scaffoldgroup such as a rigid scaffold group. The function of this scaffoldgroup is to provide molecular structure or constraint to the complex ofthe invention. In connection with a preferred connector compound of theinvention, tris-(bromomethly)benzene (TBMB), this feature may beillustrated with reference to the planar structure of the benzene groupof TBMB. This benzene group is rigid due to its planar character, andthus is able to serve as a scaffold group of the connector compound, inparticular a rigid scaffold group.

Thus in a most preferred embodiment of the invention, the connectorcompound provides conformational constraints imposed by the at leastthree covalent bonds to the polypeptide, provides further structure viathe non-covalent bonds between the connector compound and thepolypeptide, and further the connector compound of the invention alsoimposes conformational constraints by nature of its own chemicalstructure serving as a rigid scaffold. For example, the planar structureof the benzene group when the connector compound comprises same such aswhen the connector compound is tris-(bromomethly)benzene (TBMB).

Connector Compound

The connector compound is sometimes referred to as the ‘molecular core’.Suitably, the connector compound possesses molecular symmetry. Suitably,the connector compound possesses three reactive groups and possessesthreefold symmetry. This has the advantage of producing only a singlereaction product. If the connector compound is not a symmetric molecule,then multiple reaction products can be produced. This can lead tocomplications, or require that the desired isomer be separated from theother reaction products. By using a connector compound having theappropriate symmetry, such problems are advantageously ameliorated.

It is an advantage of the invention that the polypeptides produced havea greater complexity than prior art cyclic peptides. For example,polypeptides produced according to the present invention may possessmore than two loops for interaction with other chemical entities. Inaddition, polypeptides produced according to the present invention enjoya greater level of constraint than prior art based polypeptides. Thesetwo effects together create a further advantage in that multiple loops(or ‘cycles’) of the polypeptide are retained in close physicalproximity to one another via their bonds to the common connectorcompound. This provides a further level of constraint on theconformation of those polypeptides.

Typically, cyclic polypeptides of the prior art are joined usingmultiple cysteine residues such as two cysteine residues to form abridge between two parts of the peptide and thereby form a cyclicpolypeptide. However, such molecules are redox sensitive. The method ofMillward et al is directly focused at the production of cyclic peptideswhich are redox insensitive. In this regard, Millward et al's methoddeparts from the prior art and teaches away from the use of cysteines asreactive groups for the modification of polypeptides. By contrast,according to the present invention, cysteines are preferred reactivegroups.

When there are three or more reactive groups for at least three discretecovalent bonds to the connector compound, said reactive groups need noteach be cysteines. For example, the three reactive groups may compriseone cysteine and two further suitable reactive groups, which might forexample comprise lysine, selenocysteine or other(s). Most suitably allthree reactive groups are cysteines.

Prior art techniques have only led to the production of single looppolypeptides. According to the present invention, at least two loops oreven more may be produced by tethering the polypeptide at differentpoints to the connector compound.

The method of the present invention involves a minimum of three bondswith the polypeptide. This has the advantage of greater molecularconstraint. This has the further advantage of the presentation ofmultiple polypeptide loops for interaction with other moieties.

In known techniques, at best a cross linking agent has been introducedor joined to the polypeptide such as a genetically encoded polypeptide.By contrast, the present invention provides a connector compound for themultiple coordination of different parts of the same polypeptide.

Suitably the connector compound may be a small molecule. Suitably theconnector compound is a small organic molecule.

Suitably the connector compound may be, or may be based on, naturalmonomers such as nucleosides, sugars, or steroids. Suitably theconnector compound may comprise a short polymer of such entities, suchas a dimer or a trimer.

Suitably the connector compound is a compound of known toxicity,suitably of low toxicity. Examples of suitable compounds includecholesterols, nucleotides, steroids, or existing drugs such astamazapan.

Suitably the connector compound may be a macromolecule. Suitably theconnector compound is a macromolecule composed of amino acids,nucleotides or carbohydrates.

Suitably the connector compound comprises reactive groups that arecapable of reacting with functional group(s) of the target polypeptideto form covalent bonds.

The connector compound may comprise chemical groups as amines, thiols,alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters,alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkylhalides and acyl halides.

Suitably the connector compound may comprise or may consist oftris(bromomethyl)benzene or a derivative thereof.

Suitably the connector compound has a 3-fold rotational symmetry suchthat reaction of three functional groups of the target polypeptide withthe connector compound generates a single product isomer.

In some embodiments the connector compound may have a tetrahedralgeometry such that reaction of four functional groups of the encodedpolypeptide with the connector compound generates not more than twoproduct isomers.

A suitable connector compound is 1,3,5-Tris(bromomethyl)benzene(‘TBMB’).

A suitable connector compound is 2,4,6-Tris(bromomethyl)mesitylene. Itis similar to 1,3,5-Tris(bromomethyl)benzene but contains additionallythree methyl groups attached to the benzene ring. This has the advantagethat the additional methyl groups may form further contacts with thepolypeptide and hence add additional structural constraint.

The connector compound of the present invention is selected from eithera small molecule or a macromolecular structure. The said connectorcompound is composed of organic, inorganic or organic and inorganiccomponents.

In a preferred embodiment, the connector compound is a small organicmolecule as for example a linear alkane. More suitably the connectorcompound is a branched alkane, a cyclic alkane, a polycyclic alkane, anaromate, a heterocyclic alkane or a herterocyclic aromate, which offerthe advantage of being less flexible (i.e. more rigid).

In another embodiment, the connector compound is selected from amacromolecular structure as for example a polypeptide, a polynucleotideor a polysaccharide.

The connector compound of the invention contains chemical groups thatallow functional groups of the polypeptide of the encoded library of theinvention to form covalent links with the connector compound. Saidchemical groups are selected from a wide range of functionalitiesincluding amines, thiols, alcohols, ketones, aldehydes, nitriles,carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides,maleimides, azides, alkyl halides and acyl halides.

In one embodiment, the connector compound of the invention istris(bromomethyl)benzene or a derivative thereof.

Polypeptide

The functional groups of the encoded polypeptides are suitably providedby side chains of natural or non-natural amino acids. The functionalgroups of the encoded polypeptides are suitably selected from thiolgroups, amino groups, carboxyl groups, guanidinium groups, phenolicgroups or hydroxyl groups. The functional groups of the encodedpolypeptides may suitably be selected from azide, keto-carbonyl, alkyne,vinyl, or aryl halide groups. The functional groups of the encodedpolypeptides for linking to a connector compound may suitably be theamino or carboxy termini of the polypeptide.

In some embodiments each of the functional groups of the polypeptide forlinking to a connector compound are of the same type. For example, eachfunctional group may be a cysteine residue.

In some embodiments the functional groups for linking to a connectorcompound may comprise two or more different types, or may comprise threeor more different types. For example, the functional groups may comprisetwo cysteine residues and one lysine residue, or may comprise onecysteine residue, one lysine residue and one N-terminal amine.

In some embodiments, alternative amino acids such as natural amino acidsmay be suitable to chemically modify polypeptides such as phagedisplayed peptides of the invention.

Cysteine is the most suitable amino acid because it has the advantagethat its reactivity is most different from all other amino acids.Reactive groups that could be used on the connector compound to reactwith thiol groups of cysteines are alkyl halides (or also namedhalogenoalkanes or haloalkanes). Examples are bromomethylbenzene (thereactive group exemplified by TBMB) or iodoacetamide. Other reactivegroups that are used to couple selectively compounds to cysteines inproteins are maleimides. Examples of maleimides which may be used asconnector compounds in the invention include:tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene,tris-(maleimido)benzene. Selenocysteine is also a natural amino acidwhich has a similar reactivity to cysteine and can be used for the samereactions. Thus, wherever cysteine is mentioned, it is typicallyacceptable to substitute selenocysteine unless the context suggestsotherwise. Most suitably cysteine is used.

Lysines (and primary amines of the N-terminus of peptides) are alsosuited as functional groups to modify peptides on phage by linking to aconnector compound. However, they are more abundant in phage proteinsthan cysteines and there is a higher risk that phage particles mightbecome cross-linked or that they might lose their infectivity.Nevertheless, we found that lysines are especially useful inintramolecular reactions (e.g. when a connector compound is alreadylinked to the phage peptide) to form a second or consecutive linkagewith the connector compound. In this case the connector compound reactspreferentially with lysines of the displayed peptide (in particularlysines that are in close proximity). Functional groups that reactselectively with primary amines are succinimides, aldehydes or alkylhalides. Regarding alkyl halides, the reader will know that alkylhalides with different reactivities exist. In the bromomethyl group thatwe have used in a number of the accompanying examples, the electrons ofthe benzene ring can stabilize the cationic transition state. Thisparticular alkyl halide is therefore 100-1000 times more reactive thanalkyl halides that are not connected to a benzene group. Examples ofsuccinimides for use as connector compound include tris-(succinimidylaminotriacetate), 1,3,5-Benzenetriacetic acid. Examples of aldehydes foruse as connector compound include Triformylmethane. Examples of alkylhalides for use as connector compound include1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene,1,3,5-Tris(bromomethyl)benzene,1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.

In some embodiments, molecular linkers or modifications may be added to(or to create) functional groups of the encoded polypeptides beforeattachment of the connector compound wherein said linkers ormodifications are capable to react with the connector compound.

The amino acids with functional groups for linking to a connectorcompound may be located at any suitable positions within the encodedpolypeptide. In order to influence the particular structures or loopscreated, the positions of the amino acids having the functional groupsmay be varied by the skilled operator, e.g. by manipulation of thenucleic acid encoding the polypeptide in order to mutate the polypeptideproduced.

Each of the amino acids of the encoded polypeptide may be a target formutagenesis (e.g. restricted variance mutagenesis) according to theneeds of the skilled worker or the purpose to which the invention isbeing applied. Clearly at least three functional groups for bonding tothe connector compound are required on the polypeptide of interest.Amino acids other than those required for bonding to the connectorcompound may be freely varied according to operator needs and are termed‘variable amino acids’. Said variable amino acids of the encodedpolypeptide (e.g. polypeptide library member(s)) may be randomised,partially randomised, or constant.

The target polypeptide comprises a connector compound binding segment.This is the region to which the connector compound is attached. Suitablythe commentary regarding functional groups on the polypeptide is appliedto this binding segment. Suitably the connector compound binding segmentof the target polypeptide comprises 1 to 20 amino acid residues.Suitably the connector compound binding segment of the targetpolypeptide comprises fewer than 10 amino acids. This has the advantageof imposing further conformational constraint onto the polypeptidesegment when it is attached to the connector compound.

The target polypeptide suitably comprises the sequence AC(X)₆C(X)₆CG(SEQ ID No. 6), wherein X stands for a random natural amino acid, A foralanine, C for cysteine and G for glycine.

The target polypeptide suitably comprises the sequence(X)IY(X)mY(X)nY(X)o (SEQ ID No. 67), wherein Y represents an amino acidwith a functional group, X represents a random amino acid, m and n arenumbers between 1 and 20 defining the length of intervening polypeptidesegments and I and o are numbers between 0 and 20 defining the length ofthe flanking polypeptide segments.

In some embodiments, the complex of the invention may comprise apolypeptide with the sequence AC(X)₆C(X)₆CG (SEQ ID No. 6). In oneembodiment, a library member or complex of the invention may comprise amesitylene connector compound and a polypeptide with the sequenceAC(X)₆C(X)₆CG (SEQ ID No. 6), wherein the polypeptide is tethered to theexo-cyclic methyl groups of the connector compound via the cysteineresidues of the polypeptide forming three thioether bonds therewith, andwherein X stands for an amino acid, (suitably a natural amino acid), Afor alanine, C for cysteine and G for glycine.

Suitably the target polypeptide comprises an inhibitor of human plasmakallikrein and the polypeptide comprises one or more of the amino acidsequences GCSDRFRNCPADEALCG (SEQ ID No. 7), ACSDRFRNCPLWSGTCG (SEQ IDNo. 1), ACSTERRYCPIEIFPCG (SEQ ID No. 2), ACAPWRTACYEDLMWCG (SEQ ID No.3), ACGTGEGRCRVNWTPCG (SEQ ID No. 4) or a related sequence.

By related sequence is meant an amino acid sequence having at least 50%identity, suitably at least 60% identity, suitably at least 70%identity, suitably at least 80% identity, suitably at least 90%identity, suitably at least 95% identity, suitably at least 98%identity, suitably at least 99% identity. Identity is suitably judgedacross a contiguous segment of at least 10 amino acids, suitably least12 amino acids, suitably least 14 amino acids, suitably least 16 aminoacids, suitably least 17 amino acids or the full length of the referencesequence.

Suitably the target polypeptide comprises an inhibitor of humancathepsin G and the polypeptide comprises one or more of the amino acidsequences ACEYGDLWCGWDPPVCG (SEQ ID No. 8), ACIFDLGFCHNDWWNCG (SEQ IDNo. 9), ACLRAQEDCVYDRGFCG (SEQ ID No. 10) or a related sequence.

Suitably the target polypeptide comprises an inhibitor of humanurokinase-type plasminogen activator and the polypeptide comprises oneor more of the amino acid sequences ACNSRFSGCQIDLLMCG (SEQ ID No. 11),ACSRYEVDCRGRGSACG (SEQ ID No. 12) or a related sequence.

Suitably the target polypeptide is comprised by a library ofpolypeptides containing at least 10exp5 members, more suitably at least10exp9 members. The invention also relates to such libraries.

Reactive Groups of Polypeptide

The connector compound of the invention may be bonded to the polypeptidevia functional or reactive groups on the polypeptide. These aretypically formed from the side chains of particular amino acids found inthe polypeptide polymer. Such reactive groups may be a cysteine sidechain, a lysine side chain, or an N-terminal amine group or any othersuitable reactive group.

Suitably at least one functional group is a cysteine group. Groups suchas lysine or the N-terminal amines are typically not reactive enough tobond with the connector compound on their own within a convenient timeframe. However, once the connector compound has been attracted or bondedto at least one cysteine, then ordinary reaction kinetics mean that thelysine or amine bonds can rapidly and stably form thereafter. For thisreason, suitably at least one of the functional groups is a cysteinegroup.

If reactive groups on the polypeptide other than cysteine/lysine/aminegroups are desired, then a different connector compound may be chosen inorder to pair with the particular functional reactive groups of choiceon the target polypeptide.

Suitably cysteine, lysine or amine groups are used as the functional orreactive groups on the polypeptide of interest.

Suitably at least three covalent bonds are formed between the connectorcompound and the polypeptide of interest.

In some embodiments, four bonds or even more may be formed between theconnector compound and the polypeptide of interest. However, if morethan four bonds are used, then typically the product mixtures formedbecome increasingly complex and may hinder the subsequent uses orapplications. For this reason, three bonds or four bonds between theconnector compound and the polypeptide of interest are preferred. In anyembodiment, molecular symmetry of the connector compound is preferred.Most preferred are connector compounds having three functional orreactive groups. Most preferred are connector compounds having threefold molecular symmetry.

The functional groups of the genetically encoded polypeptides of theinvention are capable of forming covalent bonds to the connectorcompound/molecular core. Functional groups are specific groups of atomswithin either natural or non-natural amino acids. Preferentially,functional groups with a distinctive chemical reactivity are used tolink the polypeptide the connector compound to form the complex of theinvention. The usage of said distinctive functional groups allowsbonding of the connector compound/molecular core exclusively to thedesignated functional groups of the polypeptide but not to otherchemical groups of either the diversity elements of the polypeptide, thenucleic acid or other components of the complex.

Suitable functional groups of natural amino acids are the thiol group ofcysteine, the amino group of lysine, the carboxyl group of aspartate orglutamate, the guanidinium group of arginine, the phenolic group oftyrosine or the hydroxyl group of serine. Non-natural amino acids canprovide a wide range of functional groups including an azide, aketo-carbonyl, an alkyne, a vinyl, or an aryl halide group. The aminoand carboxyl group of the termini of the polypeptide can also serve asfunctional groups to form covalent bonds to a connectorcompound/molecular core.

The encoded polypeptides of the invention suitably contain at leastthree functional groups. Said polypeptides can also contain four or morefunctional groups. The more functional groups are used, the morediversity segments can be tethered to the connector compound/molecularcore. However, the linkage of excessive numbers of functional groups toa connector compound/molecular core is not recommended since this canlead to an unmanageable number of product isomers. Suitably three, fouror five covalent bonds to a connector compound are used; most suitablythree or four covalent bonds; most suitably three covalent bonds.

In a preferred embodiment, encoded polypeptides with three functionalgroups are generated. Reaction of said polypeptides with a connectorcompound/molecular core having a three-fold rotational symmetrygenerates a single product isomer. The generation of a single productisomer is favourable for several reasons. The nucleic acids (sometimesreferred to as the ‘genetic codes’) of the compound libraries do encodeonly the primary sequences of the polypeptide but not the isomeric stateof the molecules that are formed upon reaction of the encodedpolypeptide with the molecular core. If only one product isomer can beformed, the assignment of the nucleic acid to the product isomer isclearly defined. If multiple product isomers are formed, the nucleicacid can not give information about the nature of the product isomerthat was isolated in a screening or selection process. The formation ofa single product isomer is also advantageous if a specific member of alibrary of the invention is synthesized. In this case, the chemicalreaction of the polypeptide with the connector compound yields a singleproduct isomer rather than a mixture of isomers.

In another embodiment of the invention, encoded polypeptides with fourfunctional groups are generated. Reaction of said polypeptides with aconnector compound/molecular core having a tetrahedral symmetrygenerates two product isomers. Even thought the two different productisomers are encoded by one and the same nucleic acid (‘genetic code’),the isomeric nature of the isolated isomer can be determined bychemically synthesizing both isomers, separating the two isomers andtesting both isomers for binding to a target ligand.

In one embodiment of the invention, at least one of the functionalgroups of the polypeptides is orthogonal to the remaining functionalgroups. The use of orthogonal functional groups allows to directing saidorthogonal functional groups to specific sites of the molecular core.Linking strategies involving orthogonal functional groups may be used tolimit the number of product isomers formed. In other words, by choosingdistinct or different functional groups for one or more of the at leastthree bonds to those chosen for the remainder of the at least threebonds, a particular order of bonding or directing of specific functionalgroups of the polypeptide to specific positions on the connectorcompound may be usefully achieved.

In another embodiment, the functional groups of the encoded polypeptideof the invention are reacted with molecular linkers wherein said linkersare capable to react with a connector compound/molecular scaffold sothat the linker will intervene between the connector compound and thepolypeptide in the final bonded state.

Suitable amino acids of the members of the genetically encodedcombinatorial chemical libraries can be replaced by any natural ornon-natural amino acid. Excluded from these exchangeable amino acids arethe ones harbouring functional groups for cross-linking the polypeptidesto a molecular core. A group of adjacent amino acids that can be variedis defined as a polypeptide segment. The size of a single polypeptidesegment suitably ranges from 1 to 20 amino acids. The polypeptidesegments have either random sequences, constant sequences or sequenceswith random and constant amino acids. The amino acids with functionalgroups are either located in defined or random positions within theencoded polypeptide of the invention.

In one embodiment, the polypeptide segments that are bounded by twoamino acids harbouring functional groups for bonding with a connectorcompound/molecular core are short amino acid sequences of 10 or feweramino acids. Reaction of said encoded polypeptide sequences with amolecular core generates library members with high conformationalconstraint. Conformational constrained ligands are generally morespecific and have higher binding affinities. The conformationalconstraint can also protect the ligands from proteolytic degradation forexample in bodily fluids.

In one embodiment, an encoded polypeptide with three functional groupshas the sequence (X)IY(X)mY(X)nY(X)o (SEQ ID No. 67), wherein Yrepresents an amino acid with a functional group, X represents a randomamino acid, m and n are numbers between 1 and 20 defining the length ofintervening polypeptide segments and I and o are numbers between 0 and20 defining the length of the flanking polypeptide segments.

In a preferred embodiment, an encoded polypeptide library of theinvention has the sequence AC(X)₆C(X)₆CG (SEQ ID No. 6), wherein Arepresents alanine, C represents cysteine, X represents a random naturalamino acid and G represents glycine.

Alternatives to thiol-mediated conjugations can be used to attach theconnector compound to the peptide via covalent interactions.Alternatively these techniques may be used in modification or attachmentof further moieties (such as small molecules of interest which aredistinct from the connector compound) to the polypeptide after they havebeen selected or isolated according to the present invention—in thisembodiment then clearly the attachment need not be covalent and mayembrace non-covalent attachment. These methods may be used instead of(or in combination with) the thiol mediated methods by producing phagethat display proteins and peptides bearing unnatural amino acids withthe requisite chemical functional groups, in combination small moleculesthat bear the complementary functional group, or by incorporating theunnatural amino acids into a chemically or recombinantly synthesisedpolypeptide when the molecule is being made after theselection/isolation phase.

The unnatural amino acids incorporated into peptides and proteins onphage may include 1) a ketone functional group (as found in para or metaacetyl-phenylalanine) that can be specifically reacted with hydrazines,hydroxylamines and their derivatives (Addition of the keto functionalgroup to the genetic code of Escherichia coli. Wang L, Zhang Z, Brock A,Schultz P G. Proc Natl Acad Sci USA. 2003 Jan. 7; 100(1):56-61; BioorgMed Chem Lett. 2006 Oct. 15; 16(20):5356-9. Genetic introduction of adiketone-containing amino acid into proteins. Zeng H, Xie J, Schultz PG), 2) azides (as found in p-azido-phenylalanine) that can be reactedwith alkynes via copper catalysed “click chemistry” or strain promoted(3+2) cyloadditions to form the corresponding triazoles (Addition ofp-azido-L-phenylalanine to the genetic code of Escherichia coli. Chin JW, Santoro S W, Martin A B, King D S, Wang L, Schultz P G. J Am ChemSoc. 2002 Aug. 7; 124(31):9026-7; Adding amino acids with novelreactivity to the genetic code of Saccharomyces cerevisiae. Deiters A,Cropp T A, Mukherji M, Chin J W, Anderson J C, Schultz P G. J Am ChemSoc. 2003 Oct. 1; 125(39):11782-3), or azides that can be reacted witharyl phosphines, via a Staudinger ligation (Selective Staudingermodification of proteins containing p-azidophenylalanine. Tsao M L, TianF, Schultz P G. Chembiochem. 2005 December; 6(12):2147-9), to form thecorresponding amides, 4) Alkynes that can be reacted with azides to formthe corresponding triazole (In vivo incorporation of an alkyne intoproteins in Escherichia coli. Deiters A, Schultz P G. Bioorg Med ChemLett. 2005 Mar. 1; 15(5):1521-4), 5) Boronic acids (boronates) than canbe specifically reacted with compounds containing more than oneappropriately spaced hydroxyl group or undergo palladium mediatedcoupling with halogenated compounds (Angew Chem Int Ed Engl. 2008;47(43):8220-3. A genetically encoded boronate-containing amino acid,Brustad E, Bushey M L, Lee J W, Groff D, Liu W, Schultz P G), 6) Metalchelating amino acids, including those bearing bipyridyls, that canspecifically co-ordinate a metal ion (Angew Chem Int Ed Engl. 2007;46(48):9239-42. A genetically encoded bidentate, metal-binding aminoacid. Xie J, Liu W, Schultz P G).

Unnatural amino acids may be incorporated into proteins and peptidesdisplayed on phage by transforming E. coli with plasmids or combinationsof plasmids bearing: 1) the orthogonal aminoacyl-tRNA synthetase andtRNA that direct the incorporation of the unnatural amino acid inresponse to a codon, 2) The phage DNA or phagemid plasmid altered tocontain the selected codon at the site of unnatural amino acidincorporation (Proc Natl Acad Sci USA. 2008 Nov. 18; 105(46):17688-93.Protein evolution with an expanded genetic code. Liu C C, Mack A V, TsaoM L, Mills J H, Lee H S, Choe H, Farzan M, Schultz P G, Smider V V; Aphage display system with unnatural amino acids. Tian F, Tsao M L,Schultz P G. J Am Chem Soc. 2004 Dec. 15; 126(49):15962-3). Theorthogonal aminoacyl-tRNA synthetase and tRNA may be derived from theMethancoccus janaschii tyrosyl pair or a synthetase (Addition of aphotocrosslinking amino acid to the genetic code of Escherichia coli.Chin J W, Martin A B, King D S, Wang L, Schultz P G. Proc Natl Acad SciUSA. 2002 Aug. 20; 99(17):11020-4) and tRNA pair that naturallyincorporates pyrrolysine (Multistep engineering of pyrrolysyl-tRNAsynthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl)lysine for site-specific protein modification. Yanagisawa T, Ishii R,Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Chem Biol. 2008 Nov.24; 15(11):1187-97; Genetically encoding N(epsilon)-acetyllysine inrecombinant proteins. Neumann H, Peak-Chew S Y, Chin J W. Nat Chem Biol.2008 April; 4(4):232-4. Epub 2008 Feb. 17). The codon for incorporationmay be the amber codon (UAG) another stop codon (UGA, or UAA),alternatively it may be a four base codon. The aminoacyl-tRNA synthetaseand tRNA may be produced from existing vectors, including the pBK seriesof vectors, pSUP (Efficient incorporation of unnatural amino acids intoproteins in Escherichia coli. Ryu Y, Schultz P G. Nat Methods. 2006April; 3(4):263-5) vectors and pDULE vectors (Nat Methods. 2005 May;2(5):377-84. Photo-cross-linking interacting proteins with a geneticallyencoded benzophenone. Farrell I S, Toroney R, Hazen J L, Mehl R A, ChinJ W). The E. coli strain used will express the F′ pilus (generally via atra operon). When amber suppression is used the E. coli strain will notitself contain an active amber suppressor tRNA gene. The amino acid willbe added to the growth media, preferably at a final concentration of 1mM or greater. Efficiency of amino acid incorporation may be enhanced byusing an expression construct with an orthogonal ribosome binding siteand translating the gene with ribo-X (Evolved orthogonal ribosomesenhance the efficiency of synthetic genetic code expansion. Wang K,Neumann H, Peak-Chew S Y, Chin J W. Nat Biotechnol. 2007 July;25(7):770-7). This may allow efficient multi-site incorporation of theunnatural amino acid providing multiple sites of attachment to theligand.

Phage Purification

Any suitable means for purification of the phage may be used. Standardtechniques may be applied in the present invention. For example, phagemay be purified by filtration or by precipitation such as PEGprecipitation; phage particles may be produced and purified bypolyethylene-glycol (PEG) precipitation as described previously.

In case further guidance is needed, reference is made to Jespers et al(Protein Engineering Design and Selection 2004 17(10):709-713. Selectionof optical biosensors from chemisynthetic antibody libraries.) Suitablyphage may be purified as taught therein. The text of this publication isspecifically incorporated herein by reference for the method of phagepurification; in particular reference is made to the materials andmethods section starting part way down the right-column at page 709 ofJespers et al.

Moreover, the phage may be purified as published by Marks et al J. Mol.Biol vol 222 pp 581-597, which is specifically incorporated herein byreference for the particular description of how the phageproduction/purification is carried out.

In case any further guidance is needed, phage may be reduced andpurified as follows. Approximately 5×10¹² phage particles are reactedwith 1 mM dithiothreitol (DTT) for 30 min at room temperature, then PEGprecipitated. After rinsing with water, the pellet is resuspended in 1ml of reaction buffer (10 mM phosphate buffer, 1 mM EDTA, pH 7.8). Thephage are then optionally reacted with 50 μl of 1.6 mMN-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole(NBDIA) (Molecular Probes) for 2 h at room temperature, or more suitablyreacted with the connector compound as described herein. The reaction isterminated by PEG precipitation of phage particles.

A yet still further way in which the phage may be produced/purified isas taught in Schreier and Cortese (A fast and simple method forsequencing DNA cloned in the single-stranded bacteriophage M13. Journalof molecular biology 129(1):169-72, 1979). This publication deals withthe chain termination DNA sequencing procedure of Sanger et al. (1977),which requires single-stranded DNA as template. M13 phage DNA exists asa single strand and therefore every DNA sequence cloned in M13 can beeasily obtained in this form. Schreier and Cortese show that M13single-stranded DNA pure enough to be used as a template for sequencedetermination can be prepared by simple centrifugation of the phageparticle and extraction with phenol. The Schreier and Cortesepublication is specifically incorporated herein by reference for themethod of purification of the phage. For the avoidance of doubt, thephenol extraction is not performed for making complexes according to thepresent invention since that is for the purpose of nucleic acidpurification. Thus the phenol step of Schreier and Cortese is suitablyomitted. The Schreier and Cortese method is followed only to the pointof purified phage particles.

Thus there are myriad techniques well known in the art for purificationof phage. In the context of the present invention such purification isused for the removal of reducing agent used to reduce the functionalgroups in the polypeptide of interest for bonding to the connectorcompound, particularly when such bonding is via cysteine residues.

Optionally, especially advantageous techniques for phage purificationmay be adopted as discussed in the reaction chemistry section below. Itshould be expressly noted that these techniques are not regarded asessential for the invention, but may represent especially helpfulmethods or even the best mode of making the phage particles of theinvention. However, provided attention is paid to avoiding reoxidationof the reduced functional/reactive groups on the phage at the stage ofremoval of the reducing agent before attachment of the connectorcompound then in principle any technique may be used to accomplish this.The filtration techniques described are particularly effective but alsomore complicated than standard techniques so the operator will choosethe technique best suited to their particular working of the invention.Most suitably the filtration technique is employed.

Reaction Chemistry

In addition to the conceptual insights in connection with the triplybonded connector compound—polypeptide conjugates and phage particles ofthe invention, the inventors have also derived a precise set of chemicalconditions which can be deployed in order to achieve the chemicallinking whilst maintaining the integrity of the genetically encodedportion of the product. Prior art technologies for modification ofpolypeptides have involved harsh chemistry and independent polypeptidemodification reactions. By contrast, the present invention providesnovel chemical conditions for the modification of polypeptides whilstadvantageously retaining the function and integrity of the geneticallyencoded element of the product. Specifically, when the geneticallyencoded element is a polypeptide displayed on the surface of a phageencoding it, the chemistry advantageously does not compromise thebiological integrity of the phage. It is disclosed herein that there isa narrow window of conditions for which these chemical reactions can beenhanced or facilitated. In particular, as will be explained in moredetail below, the solvents and temperatures used are important to anefficient reaction. Furthermore, the concentration of the reagents usedare also instrumental in promoting the correct bonding, whilstameliorating or eliminating cross linking or damaging of the polypeptidemoieties which are being modified.

In particular, it is disclosed that the reduction of the cysteines inthe target polypeptide is required for the most efficient reaction.Clearly, the reducing agent used to chemically reduce those cysteinesmust be removed in order to perform the desired attachment. One knowntechnique is to use dithiothreitol (DTT) for reduction of the cysteines,and for the removal of the reducing agent to precipitate the particlessuch as the phage particles in a precipitation reaction. Suchprecipitation reactions typically involve 20% polyethylene glycol (PEG)together with 2.5 molar NaCl which leads to precipitation of the phageparticles. However, the inventors disclose that in some experimentsthese specific standard conditions did not lead to an efficient reactionof the cysteine residues in the polypeptide with the connector compound,most likely due to reoxidation of a proportion of the cysteine residueswhich had been reduced. This could not have been predicted from anunderstanding of the prior art. It should be noted that this standardtechnique may still find application in the invention, in particularwhen the skilled worker is alert to the disclosed need to be vigilant inassessing/avoiding reoxidation. However, the inventors have addressedthis cryptic problem of how to remove the reducing agent whilstmaintaining the cysteines in their reduced state. As will be disclosedin more detail below, the solutions are found in a range of strategiesincluding the use of tris carboxyethyl-phosphine, degassed buffer, theuse of chelators in the reaction mixture, and filtration in order toextract the particles under favourable chemical conditions.

Reaction conditions e.g. for attachment of the connector compound to thetarget polypeptide should be chosen carefully. Choice of conditions mayvary depending upon the application to which the invention is being put.Particular care is required when the target polypeptide is comprised bya phage particle. Guidance is provided throughout the specification andexamples section.

Reaction conditions as reaction temperature, connector compoundconcentration, solvent and/or pH should be chosen to allow efficientreaction of the functional groups of the target polypeptide with theconnector compound, but leave the nucleic acid encoding the polypeptidein a condition that allows to decode (e.g. to sequence) and/or propagatethe isolated molecules (e.g. by PCR or by phage propagation or any othersuitable technique). Moreover, the reaction conditions should leave thephage coat protein in a condition that allows it to propagate the phage.

Thiol groups of a phage encoded polypeptide may be reduced with reducingagent prior to connector compound attachment. In such embodiments, inparticular in phage display embodiments, or in particular when thereducing agent is TCEP, the excess of reducing agent is suitably removedby filtration e.g. filtration of the phage. This is especiallyadvantageous since the present inventors disclose for the first timethat conventional techniques for removal of reducing agents such asPEG/NaCl precipitation can sometimes lead to sub-optimal reaction withconnector compound, likely due to reoxidation of the reduced functionalside groups of the target polypeptide. Thus it is an advantage ofembodiments in which the target polypeptide is prepared by reductionfollowed by purification (removal of reducing agent) via filtration thatsuperior preservation of the reduced (and hence reactive) functionalgroups of the polypeptide is achieved.

In the present invention, reaction conditions are applied that on theone hand allow to efficiently link the encoded polypeptide to aconnector compound and on the other hand leave the appended nucleic acid(and phage coat proteins) in a condition that allows its propagation ordecoding. Said reaction conditions are for example the reactiontemperature, connector compound concentration, solvent composition orpH.

In one embodiment of the present invention, thiol groups of cysteineresidues are used as functional groups to link polypeptides to amolecular core. For some chemical reactions, the thiol groups of thepolypeptides need to be reduced. Thiol groups in phage displayedpolypeptides are efficiently reduced by addition of a reducing agent asfor example tris(carboxyethyl)phosphine (TCEP). Since an excess ofreducing agent can interfere with the attachment reaction it isefficiently removed by filtration of the phage.

Re-oxidation of the thiol groups after removal of TCEP is suitablyprevented by degassing of the reaction buffer.

Re-oxidation of the thiol groups is also suitably prevented by complexformation of metal ions by chelation, for example chelation withethylenediaminetetraacetic acid (EDTA).

Most suitably re-oxidation of the thiol groups is prevented or inhibitedby both chelation and use of degassed buffers.

In one embodiment of the present invention, attachment of thepolypeptide to the connector compound is accomplished by reacting thereactive groups of the polypeptide such as thiol groups of a phageencoded polypeptide with the connector compound for one hour.

Suitably they are reacted at 30° C.

Suitably they are reacted with connector compound (such astris(bromomethyl)benzene) at a concentration of 10 μM.

Suitably reaction is in aqueous buffer.

Suitably reaction is at pH 8.

Suitably reaction buffer contains acetonitrile. Suitably reaction buffercontains 20% acetonitrile.

Most suitably the reaction features two or more of the above conditions.Suitably the reaction features three or more of the above conditions.Suitably the reaction features four or more of the above conditions.Suitably the reaction features five or more of the above conditions.Suitably the reaction features six or more of the above conditions.Suitably the reaction features each of the above conditions.

These reaction conditions are optimized to quantitatively react thiolgroups of a polypeptide with the reactive groups oftris(bromomethyl)benzene. Under the same reaction conditions, about 20%of the phage particles remain infective to bring the genetic code intobacterial cells for propagation and decoding.

In one embodiment the connector compound, such as TBMB, may be attachedto the target polypeptide, such as a phage encoded polypeptide, byreaction (incubation) of thiol groups of the polypeptide for one hour at30° C. with TBMB (i.e. tris(bromomethyl)benzene) at a concentration of10 μM in aqueous buffer pH 8 containing 20% acetonitrile.

The inventors also disclose the effect of concentration of the connectorcompound in the reaction on phage infectivity. In particular theinvention suitably minimises the concentration of connector compoundused in the reaction. In other words, the lower the concentration ofconnector compound used at the time of reaction with the polypeptide ofthe phage, the better, provided always that sufficient connectorcompound becomes joined to the phage polypeptide. The advantage ofminimising the connector compound present in this way is superiorpreservation of phage infectivity following coupling of the connectorcompound. For example, when the connector compound is TBMB,concentrations of connector compound greater than 100 μM can compromiseinfectivity. Thus suitably when the connector compound is TBMB thensuitably the concentration of TBMB present at the time of bonding to thepolypeptide is less than 100 μM. Most suitably the concentration is asdisclosed in the examples section.

Post Attachment Modification

In some embodiments the polypeptide-connector compound complex may bemodified at a time following attachment.

In some embodiments, the polypeptide elements of the invention areproteolytically cleaved once they are tethered to a connectorcompound/molecular core. The cleavage generates ligands having discretepeptide fragments tethered to a connector compound/molecular core.

For example, one or more amide bonds of the polypeptide may beproteolytically cleaved after tethering the polypeptide to the molecularcore. This has the advantage of creating short polypeptides, each joinedto the connector compound by at least one covalent bond, but whichpresent different molecular structures which are retained in a complexcomprising the nucleic acid encoding the parent polypeptide. Thepolypeptide cleavage is suitably catalysed by any suitable means knownin the art such as controlled hydrolysis or more suitably enzymaticcleavage by a suitable protease. The protease may be any suitableprotease but is preferably a protease with a specific polypeptiderecognition sequence or motif. This advantageously leads to productionof more defined and/or more predictable polypeptide cleavage products.Indeed, in this embodiment, protease recognition sequences may besystematically added or removed from the target polypeptide, for exampleby manipulation of the nucleic acid(s) encoding it. This advantageouslyprovides a greater degree of control and permits greater diversity to beproduced in the molecules displayed according to the present invention.Most suitably the polypeptide comprises at least one proteaserecognition site. Suitably each said cleavage site is comprised withinamino acid sequence(s) in between functional groups on the polypeptideused for covalent bonding to the connector compound. Suitably each saidrecognition site is comprised within amino acid sequence(s) in betweenfunctional groups on the polypeptide used for covalent bonding to theconnector compound.

The peptide loops are suitably cleaved with a protease that recognizesand processes polypeptides at specific amino acid positions such astrypsin (arginine or lysine in P1 position) or thermolysin (aliphaticside chains in P1 position). The enzyme is used at a concentration thatallows efficient processing of the peptide loops of the displayedmolecule but spares the phage particle. The optimal conditions can varydepending on the length of the polypeptide loops and on the proteaseused. Trypsin for example is typically used at 200 nM in TBS-Ca buffer(25 mM Tris HCl/137 mM NaCl/1 mM CaCl₂, pH 7.4) for 10 min at 10° C. Awhole range of proteases that are suitable to modify displayedpolypeptides but that spare the phage are described in Kristensen, P.and Winter, G. (Proteolytic selection for protein folding usingfilamentous bacteriophages Fold Des. 1998; 3(5):321-8). The enzymaticprocessing of peptide on phage may be a ‘partial proteolysis’ since itcan not be excluded that a limited number of phage coat proteins arecleaved. Thus in optimisation of the conditions, the best balancebetween maximised cleavage of the target and maximum sparing of thephage particles is suitably chosen.

Suitably the target polypeptide comprises at least one such proteolyticcleavage site. Suitably the target polypeptide comprises at least twosuch proteolytic cleavage sites. Suitably the target polypeptidecomprises at least three such proteolytic cleavage sites.

In each such proteolysis embodiment, suitably the first such proteasesite occurs distal to the first covalent bond between the targetpolypeptide and the connector compound. This has the advantage that theconnector compound is retained on the complex since if the targetpolypeptide is cleaved before the first such covalent bond, then thepolypeptide-connector compound complex will be separated from thenucleic acid encoding the target polypeptide, which is undesirable forthe majority of applications of the invention.

The use of short loops (short being e.g. 6 amino acid residues or less)may compromise the ability of some proteases to cleave within the loops.In this case it may be desirable to select longer loops which are likelyto be more accessible to the protease. Furthermore after cleavage of theloops by endoprotease, it may be desirable to cut back the loops furtherwith other endoproteases, or indeed by exoproteases, such ascarboxypeptidases or aminopeptidases.

When the target polypeptide comprises more than one such protease site,suitably each of the sites occurs between two covalent bonds madebetween the target polypeptide and the connector compound. Multiplecleavage sites may occur between bonds if necessary.

In cleavage embodiments, suitably the parent polypeptide will beconsidered as a whole for the assessment of whether or not it isattached to the connector compound by at least three covalent bonds.More suitably the target polypeptide will be considered to be the intact(uncleaved) polypeptide when assessing whether or not it is attached tothe connector compound by at least three covalent bonds. Such uncleavedpolypeptides will typically be bicyclic.

Synthesis

It should be noted that once the polypeptide of interest is isolated oridentified according to the present invention, then its subsequentsynthesis may be simplified wherever possible. For example, the sequenceof the polypeptide of interest may be determined, and it may bemanufactured synthetically by standard techniques followed by reactionwith a connector compound in vitro. When this is performed, standardchemistry may be used since there is no longer any need to preserve thefunctionality or integrity of the genetically encoded carrier particle.This enables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. In this regard, largescale preparation of the candidates or leads identified by the methodsof the present invention could be accomplished using conventionalchemistry such as that disclosed in Meloen and Timberman.

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. Most suitably these steps arecarried out on the end product polypeptide/conjugate made by chemicalsynthesis, rather than on the phage.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex e.g. after theinitial isolation/identification step.

In order to illustrate the modifications/additions being described, itis helpful to consider the example of selection of a polypeptide thatreacts with a receptor. It may be desirable to extend the peptide at itsN-terminus or C-terminus. This may be useful for example in making amacrocyclic peptide that binds to one target, with a tail such as alinear tail that binds to a second target, for example a cellpenetrating peptide such as those derived from such as VP22, HIV-Tat, ahomeobox protein of Drosophila (Antennapedia) or chemically designedproteins such as polyarginine, or other such peptide e.g. as describedin (Chen and Harrison Biochemical Society Transactions (2007) Volume 35,part 4, p 821 “Cell-penetrating peptides in drug development: enablingintracellular targets”). This would have the advantage of assisting orenabling a macrocycle that had been selected against an particulartarget such as an intracellular target to enter a cell.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus using standard solid phase or solution phasechemistry. Standard protein chemistry may be used to introduce anactivatable N- or C-terminus. Alternatively additions may be made byfragment condensation or native chemical ligation e.g. as described in(Dawson P E, Muir T W, Clark-Lewis I, Kent, S B H. 1994. Synthesis ofProteins by Native Chemical Ligation. Science 266:776-779), or byenzymes, for example using subtiligase as described in (Subtiligase: atool for semisynthesis of proteins Chang T K, Jackson D Y, Burnier J P,Wells J A Proc Natl Acad Sci USA. 1994 Dec. 20; 91 (26):12544-8 or inBioorganic & Medicinal Chemistry Letters Tags for labeling proteinN-termini with subtiligase for proteomicsVolume 18, Issue 22, 15 Nov.2008, Pages 6000-6003 Tags for labeling protein N-termini withsubtiligase for proteomics Hikari A. I. Yoshihara, Sami Mahrus and JamesA. Wells).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptide to dissociate from each otheronce within the reducing environment of the cell. In this case, theconnector compound (eg. TBMB) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine could then be appended to the N-terminus ofthe first peptide, so that this cysteine only reacted with a freecysteine of the second peptide.

Similar techniques apply equally to the synthesis/coupling of twobicyclic macrocycles. Furthermore, addition of other drugs may beaccomplished in the same manner, using appropriate chemistry, couplingat the N- or C-termini or via side chains. Suitably the coupling isconducted in such a manner that it does not block the activity of eitherentity.

Thus the invention further relates to a method as described abovefurther comprising the step of extending the polypeptide at one or moreof the N-terminus or the C-terminus of the polypeptide.

Thus the invention further relates to a method as described abovefurther comprising the step of conjugating said complex or saidpolypeptide-connector compound conjugate to a further polypeptide.

Thus the invention further relates to a method as described abovewherein said conjugation is performed by

(i) appending a further cysteine to the polypeptide after bonding to theconnector compound, and

(ii) conjugating said polypeptide to said further polypeptide viadisulphide bonding to said further cysteine.

Genetically Encoded Diversity

The polypeptides of interest are suitably genetically encoded. Thisoffers the advantage of enhanced diversity together with ease ofhandling. An example of a genetically encoded polypeptide library is amRNA display library. Another example is a replicable genetic displaypackage (rgdp) library such as a phage display library. Suitably, thepolypeptides of interest are genetically encoded as a phage displaylibrary.

Thus, suitably the complex of the invention comprises a replicablegenetic display package (rgdp) such as a phage particle. In theseembodiments, suitably the nucleic acid is comprised by the phage genome.In these embodiments, suitably the polypeptide is comprised by the phagecoat.

In some embodiments, the invention may be used to produce a geneticallyencoded combinatorial library of polypeptides which are generated bytranslating a number of nucleic acids into corresponding polypeptidesand linking molecules of said connector compound to said polypeptides.

The genetically encoded combinatorial library of polypeptides may begenerated by phage display, yeast display, ribosome display, bacterialdisplay or mRNA display.

Suitably the genetically encoded combinatorial library of polypeptidesis generated by phage display. In phage display embodiments, suitablythe polypeptides are displayed on phage according to establishedtechniques such as described below. Most suitably such display isaccomplished by fusion of the target polypeptide of interest to anengineered gene permitting external display of the polypeptide ofinterest; suitably said engineered gene comprises an engineered gene 9(p9 or gene IX), gene 8 (gene VIII), gene 7 (p7 or gene VII), gene 6 (p6or gene VI) or gene 3 (p3 or gene III) of the phage. These proteinsoffer the advantage that they contain fewer or no cysteines that canreact with connector compounds such as TBMB and produce side products.For p6, it is advantageous to mutate cysteine 84 to serine. Thecysteines in p7 and p9 are most likely buried and therefore may notnecessarily need to be mutated to remove them. p8 offers the advantagethat it does not contain a cysteine residue. Thus, more suitably saidengineered gene comprises an engineered gene 8 (gene VIII), gene 6 (geneVI) or gene 3 (gene III) of the phage.

Most suitably such display is accomplished by fusion of the targetpolypeptide of interest to an engineered gene 3 protein lacking cysteineresidues in domain 1 and 2. This fusion may be accomplished by anysuitable technique known in the art such as by manipulation of thenucleic acid encoding the phage gene III protein to change the codonsencoding cysteine to codon(s) encoding other amino acid(s), and byinserting a nucleic acid sequence encoding the target polypeptide intothe gene III coding sequence in frame so that it is displayed as a geneIII fusion protein on the outside of the phage particle.

It is a benefit of the invention that the resulting engineered gene(s)leave the phage infective i.e. capable of infection and propagation.Even when the engineered gene is a gene other than gene 3, (such as gene6 or gene 8), it may still be desirable to engineer gene 3 to remove oneor more of the cysteine residue(s) (such as all of the cysteineresidues).

In a preferred embodiment, the genetically encoded polypeptides of theinvention are generated by translating a nucleic acid and linking thegenerated polypeptide to said code. The linkage of phenotype with thegenotype allows propagating or decoding the encoded ligand repertoires.Various techniques are available to link the polypeptide to itspolynucleotide code. The techniques include phage display, ribosomedisplay, mRNA display, yeast display and bacterial display and others.Encoded polypeptide repertoires comprising up to 10exp13 individualmembers have been generated with said methods. The number of individualligands that can be generated according to the invention outperformsclearly the number of individual molecules that are generally assayed inconventional screens.

In a preferred embodiment, phage display technology is used togenetically encode polypeptides of the invention. Phage display is amethod in which the gene of a polypeptide is fused to the gene of aphage coat protein. When phage are produced in a bacterial cell, thepolypeptide is expressed as a fusion of the coat protein. Upon assemblyof a phage particle the polypeptide is displayed on the surface of thephage. By contacting a phage repertoire with an immobilized antigen somephage remain bound to the antigen while others are removed by washing.The phage can be eluted and propagated. The DNA encoding the polypeptideof selected phage can be sequenced. Phage display can be used to encodemore than 10exp10 individual polypeptides. A favourable aspect of phagedisplay is that the genetic code, a single stranded DNA is packed in acoat. The coat may protect the DNA from reaction with the molecularcore.

In another preferred embodiment, the polypeptide library of theinvention is displayed on phage as a gene 3 protein fusion. Each phageparticle has about 3 to 5 copies of said phage coat protein. As a resultof the display of multiple copies of the modified polypeptide, ligandswith micromolar affinities (weak binders) can also be isolated in phageselections. Alternatively, phagemids are used to reduce the number ofpolypeptides per phage to avoid avidity effects and select ligands withhigher affinities.

In another preferred embodiment, phage with modified coat proteins areused for encoding the polypeptide libraries of the invention. Inparticular, phage lacking or having a reduced number of a specific typeof amino acid in coat proteins are used. Using said coat proteins can beadvantageous when the molecular core is reactive towards said specifictype of amino acid. This is explicitly the case when the functionalgroups of the displayed polypeptide for cross-linking a molecular coreare natural amino acids and the same type of natural amino acid ispresent at a surface exposed region in the phage coat. Using said phagewith modified coat proteins can prevent cross-linking of phage particlesthrough reaction of amino acids of multiple phage with the samemolecular core. In addition, using said phage can reduce thecross-linkage of both, amino acid side chains of the functional groupsin the polypeptide and of phage coat protein to the same molecular core.

In yet another preferred embodiment, phage with a gene 3 protein lackingthe cysteine residues of the disulfide bridges C7-C36, C46-C53,C188-C201 in domain 1 and 2 are used to display polypeptide libraries ofthe invention. A phage with mutations in said positions (C7C, C36I,C46I, C53V, C188V, C201A) and 14 additional mutations in the gene 3protein to compensate for the reduced thermal stability (T13I, N15G,R29W, N39K, G55A, 156I, I60V, T101I, Q129H, N138G, L198P, F199L, S207L,D209Y) was generated by Schmidt F. X. and co-workers (Kather, I. et al.,J. Mol. Biol., 2005). Phage without thiol groups in said minor coatprotein are suited if one or more of the functional amino acids forcross-linking the polypeptide to a molecular core are cysteine residues.Removal of the cysteine residues in the phage coat protein preventstheir interference with said bonding reaction between polypeptide andconnector compound.

This exemplary phage for application in the invention is now describedin more detail.

The disulfide-free phage of F X Schmid (domains D1-D2) comprises fdphage derived from vector fCKCBS (Krebber, C., 1997, J. Mol. Biol.). Thevector fCKCBS is based on a fd phage vector that is derived from theAmerican Type Culture Collection (ATCC: 15669-B2).

The amino acid sequence of the domains 1 and 2 of p3 of the wild-type fdphage is publicly available, for example in the PubMed database. Forease of reference, an exemplary sequence is:

(SEQ ID No. 13)AETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAPSG

F X Schmid and co-workers had evolutionarily stabilized the p3 of thisphage (Martin, A. and Schmid, F X., 2003, J. Mol. Biol.) by mutating 4amino acids. In a consecutive work F X Schmid and co-workers had mutated6 cysteines to eliminate the 3 disulfide-bridges and inserted additionalmutations to compensate for the loss of stability (Kather, I. and SchmidF X., 2005, J. Mol. Biol.). In multiple evolutionary cycles they hadgenerated clones 19, 20, 21, and 23 which have all a disulfide-free p3with varying thermal stabilities.

As explained in more detail in the examples section, the mutant 21(‘clone 21’) can be made as described, or simply obtained from F XSchmid and co-workers. According to the publication of F X Schmid thisclone contains the following mutations in the domains 1 and 2: C7S,T13I, N15G, R29W, C36I, N39K, C46I, C53V, G55A, T101I, Q129H, C188V,F199L, C201A, D209Y. In addition we found the following mutations in thedomains 1 and 2 when we sequenced the clone and compared it to wild-typefd phage: P11S and P198L. Without wishing to be bound by theory it isassumed that these mutations were already present in the phage of vectorfCKCBS.

The domains D1 and D2 of clone 21 have the following amino acidsequence:

(SEQ ID No. 14)AETVESSLAKSHIEGSFTNVWKDDKTLDWYANYEGILWKATGVVVITGDETQVYATWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYIYINPLDGTYPPGTEQNPANPNPSLEESHPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDVAFHSGFNEDLLVAEYQGQSSYLPQPPVNAPSG

The invention also relates to a library generated according to theinvention.

The invention may be applied to the screening for molecules or entitiesbinding to (or influencing binding to) a complex of the invention. Anexample of a complex of the invention is a target polypeptide with aconnector compound attached thereto. Any conventional screening formatmay be adopted by the skilled worker. The particular format used willdepend on the goals of the operator. For example, if a high throughputscreen is desired then high density, rapid turnaround and simplicity ofoperation will be paramount. Typically techniques such as phage panning,mRNA display and the like may be equally applied to the presentinvention as they are applied in the art. The key benefits of theinvention are the triple-covalent bonding of the connector compound tothe polypeptide of interest and the particular format in which theresulting complexes are screened, (or the use of those complexes ascandidate modulators of other interactions or in other screens), is amatter of choice for the person working the invention.

In one embodiment, screening may be performed by contacting a library ofthe invention with a target ligand and isolating one or more librarymember(s) that bind to said ligand.

In another embodiment, individual members of said library are contactedwith a target ligand in a screen and members of said library that bindto said target ligand are identified.

In another embodiment, members of said library are simultaneouslycontacted with a target ligand and members of said library that bind tosaid target ligand are selected.

The target ligand(s) may be a peptide, a protein, a polysaccharide, alipid, a DNA or an RNA.

The target ligand may be a receptor, a receptor ligand, an enzyme, ahormone or a cytokine.

The target ligand may be a prokaryotic protein, a eukaryotic protein, oran archeal protein. More specifically the target ligand may be amammalian protein or an insect protein or a bacterial protein or afungal protein or a viral protein.

The target ligand may be an enzyme, such as a protease. Morespecifically the target ligand may be an elastase, plasma kallikrein,cathepsin G or urokinase-type plasminogen activator.

It should be noted that the invention also embraces library member(s)isolated from a screen according to the invention. Suitably thescreening method(s) of the invention further comprise the step of:manufacturing a quantity of the ligand isolated as capable of binding tothe complex of the invention. When the screen is conducted in theopposite format (i.e. when complex(es) of the invention are identifiedby virtue of their capacity to bind to a provided ligand), suitably thescreening method(s) of the invention further comprise the step of:manufacturing a quantity of the complex of the invention isolated ascapable of binding to said ligand.

The invention also relates to library members which are, or are capableof being, isolated by a screen according to the present invention,wherein said member is subsequently generated/manufactured without thefurther use of the nucleic acid which encoded said polypeptide when partof the complex of the invention. For example, the methods of theinvention suitable further comprise the additional step of manufacturinga quantity of a polypeptide isolated or identified by a method of theinvention by attaching the connector compound to the polypeptide,wherein said polypeptide is recombinantly expressed or chemicallysynthesized. For example, when the polypeptide is recombinantlysynthesised in this embodiment, the nucleic acid originally encoding itas part of a complex of the invention may no longer be directly presentbut may have been present in an intermediate step eg. PCR amplificationor cloning of the original nucleic acid of the complex, leading toproduction of a template nucleic acid from which the polypeptide may besynthesised in this additional step.

Further Advantages

It is an advantage of the invention that the complexes themselves arecapable of propagation. Thus the complexes or libraries of the inventionmay be grown-selected-(iteratively if desired)-enriched. This contrastswith prior art techniques which require to be deconvoluted after asingle round of selection.

The present invention advantageously permits very large libraries to bebuilt and screened.

Suitably the connector compound may be flexible or rigid, more suitablythe connector compound is rigid. This has the advantage of greatermolecular constraint on the product molecule.

In some embodiments the connector compound not only constrains themolecule by holding it at three or more bonds, but also by acting as ascaffold. Amino acids of the peptide can interact with the scaffold andform a compact structure. This phenomenon may also be found inantibodies where amino acids of the CDR's interact with amino acids ofthe scaffold. Thus the invention provides this advantageous feature forthe first time on conjugated polypeptides such as those comprised byphage particles.

In some embodiments connector compounds with a symmetric geometry areused. This has the advantage that a single product is yielded ratherthan product mixtures (e.g. isomers).

Synthetic reactions have been established to link a connector compoundto a peptide via at least three covalent linkages. Prior art chemicalreaction conditions can not readily be applied to genetically encodedpeptides. We disclose a set of specific conditions which findapplication in the conjugation whilst advantageously preservinginfectivity.

It is an advantage of the invention that direct readout is obtained, inparticular for a peptide+chemical (i.e. peptide+connector compound)combination.

It is an advantage of the invention that a synthetic chemical library iscreated which is susceptible to propagation. In other words, prior arttechniques have created chemical libraries in ways in which it is notpossible to amplify/read out the small molecule of interest in thismanner i.e. even when nucleic acids have been present in the prior artchemical libraries, it has not been permissive of growth/propagation buthas rather only permitted hybridisation or other such techniques.

Similar advantages flow from the techniques described herein such as thechemical conditions used to join the connector compound to thepolypeptide which advantageously preserve infectivity of the complexwhen the complex comprises a phage particle.

Further Applications

The invention also provides a method for generating a geneticallyencoded combinatorial chemical library comprising polypeptides tetheredto a molecular core, the method comprising: (a) generating a geneticallyencoded library of polypeptides comprising functional groups capable offorming covalent bonds with a molecular core; (b) chemically linkingsaid library to said core by at least three covalent bonds.

In a broad aspect the invention relates to a polypeptide, comprising aconnector compound attached to said polypeptide, wherein the connectorcompound is attached to the polypeptide by at least three discretecovalent bonds. In particular the invention relates to such polypeptideswhich are obtainable by, or obtained by, methods of the presentinvention.

The invention may also be applied to the design and/or selection ofpeptide mimetics or small molecule mimetics for use as drugs or drugtargets.

The invention also provides methods for generating genetically encodedcombinatorial chemical libraries and for isolating ligands thereof.

The invention may be applied to identification of target hits from DNAsequencing and identifying consensus sequences in the peptides of thosetarget hits, and then synthesising the peptides. For example, aconsensus peptide may be designed by this analysis, which consensuspeptide may have an amino acid sequence not necessarily identical to anyof the hits recovered from the screening phase, and this consensuspeptide may then be synthesised according to the present invention.

The complex may comprise a phage particle.

Thus a method is provided for generating genetically encodedcombinatorial chemical libraries wherein said libraries comprisepolypeptides tethered to a molecular core via at least three covalentbonds. Libraries generated with said method are also provided.Furthermore, a method of contacting said libraries with a target ligandand isolating members that bind to said ligand is provided as arelibrary members generated with said method.

In sharp contrast to the known methods of WO 2004/077062 and WO2006/078161, the present invention provides methods for the generationand assaying of large libraries of complexes. According to WO2004/077062 and WO 2004/077062 known methods are provided to produce andscreen hundreds, or thousands of compounds. The present inventionprovides methods to genetically encode compound libraries. This allowsto generate and assay millions, billions or more individual compounds.

In contrast to the known methods of WO 2004/077062 and WO 2006/078161,the present invention provides methods to assay large compound librariesin a single reaction compartment by using in vitro selection principles.In contrast to the compounds generated according to WO 2004/077062 andWO 2006/078161, the complexes of the present invention comprise anucleic acid that allows identification of the isolated complexes;suitably said nucleic acid encodes the polypeptide of the complex.

The present invention provides reaction conditions such as connectorcompound concentration, reaction time, reaction temperature and the likethat spare the nucleic acid of the complex as for example a phageparticle, and in particular spare the infectivity of the phage particle.In other words, the chemistry presented herein preserves the function ofthe nucleic acid of the complex and preserves the biological function ofthe complex. In the example of the complex comprising a phage particlethe chemistry presented herein advantageously enhances preservedfunctionality of the phage particle and renders it possible or morepossible for it to be used in propagation of the nucleic acid aftercomplex formation.

The present invention comprises also genetically encoded combinatorialcompound libraries generated with the methods described.

The invention also relates to tricyclic polypeptides joined to aconnector compound. These may be created for example by joining the N-and C-termini of a bicyclic polypeptide joined to a connector compoundaccording to the present invention. In this manner, the joined N and Ctermini create a third loop, making a tricyclic polypeptide. Thisembodiment is suitably not carried out on phage, but is suitably carriedout on a polypeptide-connector compound conjugate of the invention.Joining the N- and C-termini is a matter of routine peptide chemistry.In case any guidance is needed, the C-terminus may be activated and/orthe N- and C-termini may be extended for example to add a cysteine toeach end and then join them by disulphide bonding. Alternatively thejoining may be accomplished by use of a linker region incorporated intothe N/C termini. Alternatively the N and C termini may be joined by aconventional peptide bond. Alternatively any other suitable means forjoining the N and C termini may be employed, for example N—C-cyclizationcould be done by standard techniques, for example as disclosed in Lindeet al. Peptide Science 90, 671-682 (2008) “Structure-activityrelationship and metabolic stability studies of backbone cyclization andN-methylation of melanocortin peptides”, or as in Hess et al. J. Med.Chem. 51, 1026-1034 (2008) “backbone cyclic peptidomimeticmelanocortin-4 receptor agonist as a novel orally administered drug leadfor treating obesity”. One advantage of such tricyclic molecules is theavoidance of proteolytic degradation of the free ends, in particular byexoprotease action. Another advantage of a tricyclic polypeptide of thisnature is that the third loop may be utilised for generally applicablefunctions such as BSA binding, cell entry or transportation effects,tagging or any other such use. It will be noted that this third loopwill not typically be available for selection (because it is notproduced on the phage but only on the polypeptide-connector compoundconjugate) and so its use for other such biological functions stilladvantageously leaves both loops 1 and 2 for selection/creation ofspecificity. Thus the invention also relates to such tricyclicpolypeptides and their manufacture and uses.

The present invention provides further methods for contacting thegenetically encoded compound libraries with a target ligand and foridentifying ligands binding to said target ligand. The geneticallyencoded compound libraries are assayed by either screening or selectionprocedures.

In a screening procedure, individual members of the library are assayed.Multiple copies of an individual member of the library are for exampleincubated with a target ligand. The target ligand is immobilized beforeor after contacting the members of the library and unbound members areremoved by washing. Bound ligands are for example detected in an enzymelinked immunosorbent assay (ELISA). The amino acid sequences of membersof the library that bind to the target ligand are determined bysequencing of the genetic code.

In a selection procedure, multiple members of the encoded compoundlibrary are contacted with a target ligand. The target ligand isimmobilized before or after contacting the members of the library andunbound members are removed by washing. The genetic code of boundligands is sequenced. Selected ligands are alternatively propagated toperform further selection rounds.

In one embodiment of the invention, the compound libraries are encodedby phage display and selections are performed by phage panning.

The target ligand of the present invention may be a protein, a DNA, aRNA or a polysaccharide. The protein can be a receptor, an enzyme, ahormone, a cytokine or a viral protein. A possible protein target ligandis a protease wherein said protease can be elastase, plasma kallikrein,cathepsin G or urokinase-type plasminogen activator.

The present invention comprises also members of the encodedcombinatorial chemical libraries isolated with methods of the invention.Said members can be produced with or without the genetic code attached.In a preferred embodiment, said members lacking the nucleic acid areused as drug or drug lead.

Several members of the encoded combinatorial chemical libraries that arecapable of binding to a target ligand were isolated with a method of thepresent invention. Said members are composed of a mesitylene core and apolypeptide with the sequence AC(X)₆C(X)₆CG (SEQ ID No. 6), wherein thepolypeptide is tethered to the exo-cyclic methyl groups of the core viathe cysteine residues forming three thioether bonds and wherein X standsfor a natural amino acid, A for alanine, C for cysteine and G forglycine. The peptide portion of said members can be expressedrecombinantly or be synthesized chemically.

The present invention provides inhibitors of human plasma kallikreinisolated with methods of the invention from encoded combinatorialchemical libraries of the invention. Said inhibitors have either of thepolypeptide sequences GCSDRFRNCPADEALCG (SEQ ID No. 7),ACSDRFRNCPLWSGTCG (SEQ ID No. 1), ACSTERRYCPIEIFPCG (SEQ ID No. 2),ACAPWRTACYEDLMWCG (SEQ ID No. 3), ACGTGEGRCRVNWTPCG (SEQ ID No. 4) orrelated sequences wherein the thiol groups of the cysteines are linkedto mesitylene cores.

The present invention provides also inhibitors of human cathepsin Gisolated with methods of the invention form encoded combinatorialchemical libraries of the invention. Said inhibitors have either of thepolypeptide sequences ACEYGDLWCGWDPPVCG (SEQ ID No. 8),ACIFDLGFCHNDWWNCG (SEQ ID No. 9), ACLRAQEDCVYDRGFCG (SEQ ID No. 10) orrelated sequences wherein the thiol groups of the cysteines are linkedto mesitylene cores.

The present invention provides also inhibitors of human urokinase-typeplasminogen activator isolated with methods of the invention formencoded combinatorial chemical libraries of the invention. Saidinhibitors have either of the polypeptide sequences ACNSRFSGCQIDLLMCG(SEQ ID No. 11), ACSRYEVDCRGRGSACG (SEQ ID No. 12) or related sequenceswherein the thiol groups of the cysteines are linked to mesitylenecores.

Biological Targets

It is important to create and assay as many molecules as possible sincethe chance to identify a ligand with desired properties increases whenmore molecules are tested. Also, in general, ligands with higheraffinities are obtained when larger molecule repertoires are assayed.

Researchers typically evaluate molecules using screening or selectionmethodologies. Screening is a process by which compounds areindividually assayed for their ability to modify a target. Screeningprocesses are versatile and allow the assaying of molecule repertoireshaving a manifold of structures. Screening by individual assays,however, may be time-consuming and the number of unique molecules thatcan be tested for binding to a specific target generally does not exceed10exp6 chemical entities. In contrast, selection methods generally allowthe sampling of a much larger number of different molecules. Thusselection methods are more suitably used in application of theinvention. In selection procedures, molecules are assayed in a singlereaction vessel and the ones with favourable properties (i.e. binding)are physically separated from inactive molecules. Selection strategiesare available that allow to generate and assay simultaneously more than10exp13 individual compounds. Examples for powerful affinity selectiontechniques are phage display, ribosome display, mRNA display, yeastdisplay, bacterial display or RNA/DNA aptamer methods. These biologicalin vitro selection methods have in common that ligand repertoires areencoded by DNA or RNA. They allow the propagation and the identificationof selected ligands by sequencing. Phage display technology has forexample been used for the isolation of antibodies with very high bindingaffinities to virtually any target.

INDUSTRIAL APPLICATION

The present invention is applicable to the discovery of molecules thatare useful in the fields of biology, biotechnology and pharmaceuticalsciences. In particular the present invention relates to methods for thegeneration of drugs or drug leads.

The present invention comprises methods for the generation ofgenetically encoded combinatorial chemical libraries and methods for theisolation of members of said libraries. Furthermore, the inventioncomprises libraries generated with said methods and members of thelibraries isolated with said methods.

The present invention provides a method for the generation ofgenetically encoded combinatorial chemical libraries wherein the membersof said libraries comprise a central molecular core and multiplediversity elements that are appended to said core. Said geneticallyencoded libraries are generated in two steps: In the first step,genetically encoded polypeptide libraries comprising functional groupscapable of forming covalent bonds with a molecular core are generated.In the second step, the genetically encoded polypeptide libraries arechemically cross-linked to said molecular core by at least threecovalent bonds.

Molecules of the genetically encoded combinatorial chemical libraries ofthe present invention have a core structure that is expanded by variousappendages. Unlike state of the art genetically encoded combinatorialchemical libraries generated with biological methods, the libraries ofthe present invention provide molecules with non-linear, branchedarchitectures. Molecules with such branched structures are suitable forbinding to target ligands since they can bind to the target throughinteraction of multiple appendages that point away from a central core.

In contrast to linear polymeric structures, the complexes of the presentinvention have less conformational flexibility. In solution they adoptonly a limited set of conformations. As a consequence, binding of saidcomplexes or polypeptides to a target ligand is not associated with adramatic loss of entropy and high binding affinities can result.

The polypeptides of the complexes/libraries of the invention aregenetically encoded. This allows very powerful biological encodingmethods as for example phage display, ribosome display, yeast display,bacterial display or mRNA display can be applied for their productionwhich allows to generate ligand libraries containing millions, billionsor more individual members.

The sequences of the polypeptide appendages of members of thegenetically encoded combinatorial chemical libraries can be varied.Exceptions are amino acids that harbour functional groups forcross-linking the polypeptide to a molecular core, which is explained inmore detail herein. The polypeptide appendages can comprise very largecombinatorial diversities. Representing large combinatorial repertoiresis important since the probability of isolating high-affinity binders totarget ligands increases with library size.

Unlike linear biopolymers as polypeptides, DNA or RNA aptamers, thecomplexes and members of the genetically encoded combinatorial chemicallibraries of the invention do not form complex tertiary structures. Thecomplexes and members of the genetically encoded combinatorial chemicallibraries of the invention enjoy greatly reduced risk of inactivationthrough irreversible unfolding. The formation of aggregates due tounfolding is thus advantageously unlikely.

The genetically encoded libraries of the invention suitably comprise atleast 10exp5 individual members. Preferentially, said libraries comprisemillions or billions or more of individual members. The size of saidlibraries is determined by the methods that are used to link the nucleicacid encoding a polypeptide with that polypeptide. In a preferredembodiment of the invention, biological methods are used to generategenetically encoded polypeptide repertoires. The number of individualmembers of polypeptides that are linked to the encoding polynucleotidecode may exceed 10exp13 depending on the methods used.

EXAMPLES

Overview

In these examples we demonstrate manufacture of phage encodedcombinatorial chemical libraries.

The discovery of synthetic molecules with high affinity and specificityfor biological targets is a central problem in drug discovery. While itbecame recently possible to isolate large molecular structures asantibodies or aptamers to virtually any target using in vitro selectiontechniques, the generation of small organic binders with high affinitiesremained a great challenge. In this invention, we disclose a strategyfor the isolation of small molecule structures that are built of anorganic molecule core (connector compound) that is decorated withpeptidic moieties (e.g. polypeptide(s)). For convenience in theseexamples, phage display technology was used to encode the peptidefraction of the small molecules allowing the generation and selection ofvery large combinatorial chemical repertoires. Reaction conditions werechosen to selectively tether a 17 amino acid peptide via three thioetherbonds to a benzene molecule but spare the coat proteins of the phageparticles. Highly specific binders with sub-micromolar affinities wereobtained against the two human serine proteases plasma kallikrein andcathepsin G. An affinity maturated inhibitor of human plasma kallikreinwith an apparent K_(i) of 1.5 nM efficiently suppressed contactactivation in human plasma.

Background to the Examples

Molecules with high affinity and specificity for biological targets areneeded to develop efficient and selective therapies against a wide rangeof diseases. The process of finding a new small organic molecule drugagainst a chosen target usually involves high-throughput screening,wherein large libraries of chemicals are tested for their ability tomodify the target. The process, however, is time-consuming and costlyand the number of unique molecules that can be tested against a specifictarget generally does not exceed a million chemical entities. Thescreens often only provide leads, which then require further improvementeither by empirical methods or by chemical design. More powerful methodsfor the generation of binding molecules are biological in vitroselection techniques as phage display, ribosome display, mRNA display orRNA/DNA aptamer techniques. They allow the rapid generation of largecombinatorial repertoires (10⁹-10¹³) of polypeptides, RNA or DNA and thesubsequent isolation of binders with high affinities. However, therestriction of such methods to large biopolymer structures as antibodiesor aptamers precludes their use for small-molecule discovery.

In order to apply in vitro selection to combinatorial chemistrylibraries, various methodologies have been proposed to associate organicmolecules with a tag that specifies its structure. Most approachesproposed the use of DNA tags to identify the small organic moleculesafter affinity selection. A process of parallel combinatorial synthesisto encode individual members of a large library of chemicals with uniquenucleotide sequences on beads has been proposed (Brenner, S. and Lerner,R. A., PNAS, 1992). After the chemical entity is bound to the target,the genetic code is decoded by sequencing of the nucleotide tag. A smallcollection of organic molecules has been conjugated to DNAoligonucleotides and performed affinity selections with differentantigens (Doyon, J. B. et al., JACS, 2003). Neri D. and co-workers hadgenerated large repertoires of molecule pairs by self-assembly ofsmaller DNA encoded chemical sub-libraries through hybridization of twoDNA strands (Melkko, S. et al., Nature Biotechnol., 2004). Themethodology was successfully used for affinity maturation of smallmolecule ligands. Halpin D. R. and Harris P. B. developed a strategy forthe in vitro evolution of combinatorial chemical libraries that involvesamplification of selected compounds to perform multiple selection rounds(Halpin, D. R. and Harbury, P. B., PLOS Biology, 2004). Woiwode T. F. etal. attached libraries of synthetic compounds to coat proteins ofbacteriophage particles such that the identity of the chemical structureis encoded in the genome of the phage (Woiwode, T. F., Chem. & Biol.,2003). All these strategies employing DNA encoded chemical compoundshave proven to be efficient in model experiments and some have evenyielded novel small molecule binders. However, it became apparent thatthe encoding of large compound libraries and the amplification ofselected compounds is much more demanding than the equivalent proceduresin biological selection systems.

In this invention we teach a strategy for encoding hybrid peptide-smallmolecule structures that are built of multiple polypeptide fragmentstethered to a central small organic molecule. The peptide portion isencoded by phage particles allowing the generation and selection of verylarge and complex diversities. We envisioned the following reactionprocedures to link peptide fragments to a small molecule (schematicallydepicted in FIG. 1). A chemical structure equipped with reactive groupsis incubated with a phage displayed peptide. Specific amino acids in thepeptide (e.g. cysteines) react with the functional groups of the smallmolecule to form covalent bonds wherein a first linkage acceleratesconsecutive linkages. The resulting molecules are then subjected toaffinity selections. Alternatively, specific peptide bonds of themulti-cyclic structure are enzymatically cleaved to obtain smallchemical structures decorated with discrete peptide entities. Theattachment of phage displayed polypeptide repertoires to small molecularstructures is not trivial as the reaction needs to be specific andselective to yield a single product. Also, the reactants suitably shouldnot impair the phage particle. Furthermore, linking a small molecule viamultiple sites to a peptide adds an additional level of complexity asproduct mixtures could easily be generated or phage particles could becross-linked. In fact no example is known in the art where a smallmolecule was linked to a polypeptide displayed on phage via more thanone bond.

Materials and Methods

Chemical Linkage of Peptide-D12 Fusion Proteins to a Chemical Scaffold

The domains D1-D2 of the g3p (comprising amino acid residues 2 to 217 ofthe mature fdg3p) with and without the N-terminally fused peptide^(N)ACGSGCGSGCG^(C) (SEQ ID No. 16) was expressed in E. coli. The pUC119based expression vector with a leader sequence and the D1-D2 gene with aC-terminal hexa-histidine tag (here termed pUC119H6D12) was kindlyprovided by Phil Holliger from the laboratory of molecular biology (LMB)in Cambridge. A plasmid for expression of D1-D2 with the N-terminalpeptide was cloned by PCR amplification of the D1-D2 gene with theprimers pepd12ba (encoding the peptide sequence) and d12fo and ligationinto the SfiI/NotI digested pUC119H6D12. The gene for expression ofdisulfide-free D1-D2 with a total of 20 amino acid mutations was kindlyprovided by Insa Kather and Franz Xaver Schmid from the University ofBayreuth. The gene was PCR amplified from the vector fdg3p0ss21 witheither the primer pair d120ssba/d120ssfo, pepd120ssba/d120ssfo,P2cd120ssba/d120ssfo or P1cd120ssba/d120ssfo and SfiI/NotI ligated intopUC119H6D12 for expression of disulfide-free D1-D2 with and without theN-terminal fused peptides ^(N)ACGSGCGSGCG^(C) (SEQ ID No. 16),^(N)AGSGCGSGCG^(C) (SEQ ID No. 17) or ^(N)AGSGKGSGCG^(C) (SEQ ID No.18). All 6 proteins were expressed in TG1 E. coli cells at 30° C. for 8hours and the periplasmic fraction was purified stepwise by Ni-affinitychromatography and gel filtration on a Superdex 75 column in 20 mMNH₄HCO₃ pH 7.4.

Oxidized sulfhydryl groups were reduced by incubation of the protein(1-10 μM) with 1 mM TCEP in 20 mM NH₄HCO₃, pH 8 at 42° C. for 1 hr. Thereducing agent was removed on a vivaspin 20 filter having a MWCO of10,000 (Vivascience, Stonehouse, UK) using 20 mM NH₄HCO₃, 5 mM EDTA, pH8 buffer. The thiol groups of the proteins were reacted by incubationwith 10 μM TBMB in reaction buffer (20 mM NH₄HCO₃, 5 mM EDTA, pH 8, 20%ACN) at 30° C. for 1 hr. For removal of non-reacted TBMB andconcentration the protein was filtered with a microcon YM-30 (Millipore,Bedford, Mass.). The molecular masses of the proteins (5-20 M) weredetermined by denaturing in 4 volumes of 50% MeOH, 1% formic acid andanalysis on a time of flight mass spectrometer with electrosprayionization (Micromass, Milford, Mass., USA). Molecular masses wereobtained by deconvoluting multiply charged protein mass spectra usingMassLynx version 4.1. The performance of the chemical modificationreaction in presence of phage was tested by addition of PEG purifiedphage to a final concentration of 10¹⁰ t.u. to the protein before TCEPreduction. The phage was removed by gel filtration with a PD-10 column(Amersham Pharmacia, Uppsala, Sweden) after TBMB reaction.

Creation of a Phage Peptide Library

The genes encoding a semi-random peptide with the sequenceAla-Cys-(Xaa)₆-Cys-(Xaa)₆-Cys (SEQ ID No. 19), the linkerGly-Gly-Ser-Gly (SEQ ID No. 20) and the two disulfide-free domains D1and D2 were cloned in the correct orientation into the phage vectorfd0D12 to obtain phage library 1. The vector fd0D12, lacking the genesof the D1 and D2 domains of gene 3 and having a second SfiI restrictionsite was previously created by whole-plasmid PCR amplification offdg3p0ss21 (Kather, I. et al., J. Mol. Biol., 2005) using the primerecoG3pNba and pelbsfiecofo. The genes encoding the peptide repertoireand the two gene 3 domains were step-wise created in two consecutive PCRreactions. First, the genes of D1 and D2 were PCR amplified with the twoprimer preper and sfi2fo using the vector fdg3p0ss21 as a template.Second, the DNA encoding the random peptides was appended in a PCRreaction using the primer sficx6ba and sfi2fo. The ligation of 33 and 9μg of SfiI digested fd0D12 plasmid and PCR product yielded 4.4×10⁹colonies on 12 20×20 cm chloramphenicol (30 μg/ml) 2YT plates. Colonieswere scraped off the plates with 2YT media, supplemented with 15%glycerol and stored at −80° C. Glycerol stocks were diluted to OD₆₀₀=0.1in 1 liter 2YT/chloramphenicol (30 μg/ml) cultures and phage wereexpressed at 30° C. over night (12 to 16 hrs).

Chemical Linkage of a Phage Displayed Peptide to a Small Molecule

Typically 10¹¹-10¹² t.u. of PEG purified phage were reduced in 20 ml of20 mM NH₄HCO₃, pH 8 with 1 mM TCEP at 42° C. for 1 hr. The phage werespun at 4000 rpm in a vivaspin-20 filter (MWCO of 10,000) to reduce thevolume to 1 ml and washed twice with 10 ml ice cold reaction buffer (20mM NH₄HCO₃, 5 mM EDTA, pH 8). The volume of the reduced phage wasadjusted to 32 ml with reaction buffer and 8 ml of 50 μM TBMB in ACNwere added to obtain a final concentration of 10 μM. The reaction wasincubated at 30° C. for 1 hr before non-reacted TBMB was removed byprecipitation of the phage with ⅕ volume of 20% PEG, 2.5 M NaCl on iceand centrifugation at 4000 rpm for 30 minutes.

Phage Selections with Human Plasma Kallikrein and Cathepsin G

Human plasma kallikrein (activated with factor XIIa) was purchased fromInnovative Research (Southfield, Mich., USA) and biotinylated at aconcentration of 1.2 μM with a 5-fold molar excess ofSulfo-NHS-LC-biotin (Pierce, Rockford, Ill., USA) in PBS, pH 7.4/5% DMSOat RT for 1 hr. The biotinylated protein was purified on a PD-10 columnusing a buffer of 50 mM NaAc, pH 5.5, 200 mM NaCl. Readily biotinylatedhuman cathepsin G was purchased from Lee Biosolutions (St. Louis, Mich.,USA). Biotinylated antigens (5 to 20 μg) were incubated with 50 μlmagnetic steptavidin beads (Dynal, M-280 from Invitrogen, Paisley, UK)for 20 minutes at 4° C. The antigen-coated beads were washed twice withwashing buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mMCaCl₂) and blocked in 0.5 ml washing buffer containing 1% BSA and 0.1%tween 20 for 30 minutes. The chemically modified phage (typically10¹⁰-10¹¹ t.u. dissolved in 2 ml washing buffer) were blocked byaddition of 1 ml of washing buffer containing 3% BSA and 0.3% tween 20.3 ml blocked phage were pipetted to 0.5 ml blocked magnetic beads andincubated on a rotating wheel at RT. The beads were washed 8 times withwashing buffer containing 0.1% tween 20 and twice with washing bufferbefore incubation with 100 μl of 50 μM glycine, pH 2.2 for 5 minutes.Eluted phage were transferred to 50 μl of 1 M Tris-Cl, pH 8 forneutralization, incubated with 50 ml TG1 cells at OD₆₀₀=0.4 for 90minutes at 37° C. and the cells were plated on large 2YT/chloramphenicolplates. Two additional rounds of panning were performed using the sameprocedures. In the second round of selection, neutravidin-coatedmagnetic beads were used to prevent the enrichment ofstreptavidin-specific peptides. The neutravidin beads were prepared byreacting 0.8 mg neutravidin (Pierce, Rockford, Ill., USA) with 0.5 mltosyl-activated magnetic beads (Dynal, M-280 from Invitrogen, Paisley,UK) according to the supplier's instructions.

Screening Procedure to Identify Protease Inhibitors

The plasmid DNA of clones selected after the second and third rounds ofbiopanning was PCR-amplified in a single tube with the primer 21seqbaand flagfo and cloned into the vector pUC119H6D12 at the SfiI and NotIsites for the periplasmic expression of the peptides fused to thedisulfide-free D1 and D2 domains with a C-terminal FLAG tag and ahexa-histidine-tag. The ligated plasmids were electroporated into TG1cells and plated on 2YT/ampicillin (100 μg/ml) plates. Clones expressingthe recombinant protein were identified as follows: 1 ml cultures of2YT/ampicillin (100 μg/ml) in 96-deep well plates were inoculated withcells of individual colonies and incubated at 37° C. Protein expressionwas induced with 1 mM IPTG when the cultures were turbid and the plateswere shaken 300 rpm at 30° C. o/n. The cells were pelleted bycentrifugation at 3500 rpm for 30 minutes, lysed with washing buffercontaining 1 mg/ml lysozyme and spun at 3500 rpm to pellet the celldebris. The supernatants were transferred to 96-well polysorp plates(Nunc, Roskilde, Denmark) for non-specific adsorbtion. The wells wererinsed twice with washing buffer containing 0.1% tween 20 and blockedwith washing buffer containing 1% BSA and 0.1% tween 20 for 1 hr.Anti-FLAG M2-peroxidase conjugate (Sigma-Aldrich, St. Louis, Mo., USA)was 1:5000 diluted and blocked in washing buffer containing 1% BSA and0.1% tween 20 and added to the plates for 1 hr. The wells were washed (5times with washing buffer containing 0.1% tween 20 and once withoutdetergent) and the bound peroxidase was detected with TMB substratesolution (eBiosciences, San Diego, USA). The plasmid DNA of proteinexpressing clones was sequenced (Geneservice, Cambridge, UK). Selectedclones were expressed on an 800 ml scale and purified by Ni-affinitychromatography and gel filtration as described above. The peptides werechemically modified using the procedure described above and theconcentrations of the products were determined by measuring the opticalabsorption at 280 nm. The IC₅₀ was measured by incubating variousconcentrations of the modified peptide fusion proteins (2-folddilutions) with human plasma kallikrein (0.1 nM) or cathepsin G (20 nM)and determining the residual activity in 10 mM Tris-Cl, pH 7.4, 150 mMNaCl, 10 mM MgCl₂, 1 mM CaCl₂, 0.1% BSA, 0.01% triton-X100. Human plasmakallikrein activity was measured with the fluorogenic substrateZ-Phe-Arg-AMC (Bachem, Bubendorf, Switzerland) at a concentration of 100μM on a Spectramax Gemini fluorescence plate reader (excitation at 355nm, emission recording at 460 nm; Molecular Devices, Sunnyvale, Calif.,USA). Human cathepsin G activity was measured with the colorimetricsubstrate N-Suc-Ala-Ala-Phe-Pro-(SEQ ID No. 21) pNA (Bachem, Bubendorf,Switzerland) at a concentration of 1 mM with a Spectramax absorptionplate reader (recording at 410 nm; Molecular Devices, Sunnyvale, Calif.,USA).

Phage Selections for Affinity Maturation of Human Plasma KallikreinInhibitors

Three peptide phage libraries were created essentially as the library 1(see above) but using the degenerate primer sficx6abc (library 2),sficx6abb (library 3) and sficx6aba (library 4) instead of sficx6ba.Electroporation of the ligation reactions into TG1 cells yielded 9.5×10⁸(library 2), 1.1×10⁹ (library 3) and 1.2×10⁹ (library 4) transformants.Phage of each library were produced in 1 L cultures, purified, pooledand reacted with TBMB. Three rounds of panning were performedessentially as in the selections described above but using thebiotinylated human plasma kallikrein at a lower concentration (1 nM inthe 1^(st) and 2^(nd) rounds, 200 pM in the 3^(rd) round).

Chemical Synthesis of Bicyclic Peptides

Peptides with a free amine at the N-terminus and an amide at theC-terminus were chemically synthesized on a 25 mg scale by solid phasechemistry (JPT Peptide Technologies, Berlin, Germany). The crudepeptides in 1 ml 60% NH₄HCO₃, pH 8 and 30% ACN (1 mM) were reacted withTBMB (1.2 mM) for 1 hr at RT. The reaction product was purified byreversed-phase high-performance liquid chromatographic (HPLC) using aC18 column and gradient elution with a mobile phase composed of ACN and0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 2ml/min. The purified peptides were freeze-dried and dissolved in DMSO ora buffer of 50 mM Tris-Cl pH 7.8, 150 mM NaCl for activity measurements.

Activity and Specificity Measurement of Human Plasma KallikreinInhibitors

Inhibitory activities (IC₅₀) were determined by measuring residualactivities of the enzyme upon incubation (30 min, RT) with differentconcentrations of inhibitor (typically ranging from 10 μM to 0.5 pM).The activities of human plasma kallikrein (0.1 nM) and factor XIa (0.8nM; Innovative Research, Southfield, Mich., USA) were measured withZ-Phe-Arg-AMC (100 μM) and the activity of human thrombin (2 nM;Innovative Research, Southfield, Mich., USA) with Boc-Phe-Ser-Arg-AMC(100 μM) in 10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM CaCl₂,0.1% BSA, 0.01% triton X-100 and 5% DMSO. Recombinant mouse plasmakallikrein from R&D Systems (Minneapolis, Minn., USA) with a signalpeptide was proteolytically activated with 0.5 mg/ml thermolysin at 37°C. for 1 hr. The activity of mouse plasma kallikrein (3 nM) was measuredwith Z-Phe-Arg-AMC (100 μM) in 50 mM Tris-Cl pH 7.5, 10 mM CaCl₂ and 250mM NaCl, 0.1% BSA, 0.01% triton X-100 and 5% DMSO. Inhibitor hydrolysedin one binding loop was generated by incubation of TBMB modified peptidePK15 with human plasma kallikrein at a molar ration of 5:1 for 24 hoursat 37° C. and subsequent heat inactivation of the enzyme at 60° C. (30min). Apparent K_(i) values were calculated according to the Cheng andPrusoff equation (Cheng, Y. and Prusoff, W. H., Biochem. Pharmacol.,1973).

Measurement of Contact Activation in Human Plasma

Normal human plasma from single donors was purchased from 3H Biomedical(Uppsala, Sweden). The plasma was centrifuged at 1500×g at 20° C. for 15minutes to obtain platelet-poor plasma (PPP). Aliquots of the PPP werestored in polypropylene tubes at −80° C. Samples of 60 μl PPP containing5, 50, 500 or 5000 nM of aprotinin (Roche, Mannheim, Germany) or TBMBmodified peptide PK15 were prepared. The thrombin activation time wasmeasured at 37° C. by addition of 20 μl of 1:10 diluted actin FS (DadeBehring, Marburg, Germany) and 20 μl of 20 mM hepes buffer pH 7.4, 100mM CaCl₂, 50 mg/ml BSA and 1 mM Z-Gly-Gly-Arg (SEQ ID No. 22)-AMC to theplasma sample and monitoring of the fluorescence intensity with afluorescence plate reader (excitation at 355 nm, emission recording at460 nm; PHERAStar, Labtech, Offenburg, Germany). Activation of factorXIIa and human plasma kallikrein were measured as follows. 2 μg kaolinwas added to the plasma samples, mixed well and incubated for 20 minutesat 37° C. The samples were diluted 250-fold in 50 mM Tris-Cl pH 7.8, 150mM NaCl. Plasma kallikrein-like activity was measured with thechromogenic substrate H-D-Pro-Phe-Arg-pNA (100 μM; Bachem, Bubendorf,Switzerland) using an absorption plate reader (absorption at 450 nm;Molecular Devices, Sunnyvale, Calif., USA).

Structure Determination of TBMB Modified Peptide PK15

1 mg of TBMB modified peptide PK15 was dissolved in 550 μl 10 mMdeuterated Tris HCl pH 6.6, 150 mM NaCl, 10 mM MgCl₂, 1 mM CaCl₂ toobtain an inhibitor concentration of 1 mM. Spectra of the inhibitor wererecorded at 800 MHz (Bruker Avance with TCI cryoprobe). Spectralassignments were based on TOCSY and NOESY spectra. Distance restraintswere from the NOESY spectra. 50 structure conformers were calculated.The program PyMOL was used for structure analysis and visualization ofthe molecular models.

Example 1 Making a Complex

In this example we demonstrate attaching phage displayed peptides tosmall molecules. The polypeptide in this example is a phage displayedpeptide. The nucleic acid is comprised by the phage particle. Theconnector compound of this example is a small molecule (TBMB in thisexample).

We used the small organic compound tris-(bromomethyl)benzene (TBMB) as ascaffold to anchor peptides containing three cysteine residues (Kemp, D.S. and McNamara, P. E., J. Org. Chem, 1985; FIG. 1B). Halogene alkanesconjugated to an aromatic scaffold react specifically with thiol groupsof cysteines in aqueous solvent at room temperature (Stefanova, H. I.,Biochemistry, 1993). Meloen and co-workers had previously usedbromomethyl-substituted synthetic scaffolds for the immobilization ofpeptides with multiple cysteines (Timmerman, P. et al., ChemBioChem,2005). The mild conditions needed for the substitution reaction areconvenient to spare the functionality of the phage (Olofsson, L., etal., J. of Molecular Recognition, 1998). We chose cysteines as anchoringpoints because their side chains have the most distinguished reactivitywithin the 20 natural amino acids. Also, cysteine residues are rare inproteins of the phage coat (8 cysteines in pill, one cysteine in pVI,pVII and pIX; Petrenko, V. A. and Smith, G. P., Phage Display inBiotechnology and Drug Discovery, 2005). The three-fold rotationalsymmetry of the TBMB molecule ensures the formation of a uniquestructural and spatial isomer upon reaction with three cysteines in apeptide.

The reaction conditions for the modification of a peptide on phage wereelaborated next. As it appeared difficult to detect the chemicallymodified peptide on phage with available techniques, we expressed thepeptide ^(N)GCGSGCGSGCG^(C) (SEQ ID No. 15) as an N-terminal fusion withthe two soluble domains D1 and D2 of the minor phage coat protein pilland analyzed the molecular weight of the protein before and afterreaction with TBMB by mass spectrometry. Attempts to selectively linkthe three cysteines in the peptide to the scaffold but spare the threedisulfide bridges of the D1 and D2 domains of pill (C7-C36, C46-C53,C188-C201) failed. This prompted us to take advantage of adisulfide-free gene-3-protein recently developed by Schmidt F. X. andco-workers (Kather, I. et al., J. Mol. Biol., 2005). The peptide fusedto the N-terminal domain of the cysteine-free gill protein was reducedwith tris(carboxyethyl)phosphine (TCEP). As the reducing agent was foundto react with the bromomethyl groups of the TBMB scaffold, it wasremoved before the addition of TBMB to the protein. Re-oxidation of thethiol groups after removal of TCEP could be prevented by degassing ofthe reaction buffer and complexation of metal ions with 5 mM EDTA.Reaction of the thiol groups with TBMB at various concentrations andmass spectrometric analysis of the product revealed that a concentrationof 10 μM TBMB is sufficient for quantitative modification of the peptideat 30° C. in one hour. Predominantly one product was formed with theexpected molecular mass (Δ mass expected=114 Da; FIG. 2A). When thedisulfide-free D1-D2 without a fused peptide was incubated with TBMB,its mass was not changed indicating that non-specific reactions withother amino acids do not occur. Addition of phage particles to thereactions (10¹⁰ t.u. per milliliter) revealed that the high density ofphage coat proteins in the vessel does not encumber the reaction of thepeptide with TBMB. Unexpectedly, we found that reaction of TBMB withpeptides containing only two cysteine residues (^(N)AGSGCGSGCG^(C) (SEQID No. 17)-D1-D2) yields a product with a molecular mass that isconsistent with the reaction of the remaining bromomethyl group with theprimary amine of the N-terminus (FIGS. 7A and 7B). Similarly, thereaction of TBMB with a peptide having one cysteine and a lysine(^(N)AGSGKGSGCG^(C) (SEQ ID No. 18)-D1-D2) yields a molecular mass thatis expected when the primary amines of lysine and the N-terminus reactwith the remaining two bromomethyl groups (FIGS. 7C and 7D).

Next, we tested whether phage modified with TBMB were still able toinfect bacteria. We found that the higher the TBMB concentration in thereaction was, the fewer phage remained infective (FIG. 2B). At reactionconditions that allow the quantitative modification of the peptide (10μM TBMB, 30° C., reaction for 1 hour) the number of infective phagedropped by a factor 5.

Example 2 Screening

This example shows affinity selection of inhibitors for human plasmakallikrein and cathepsin G.

The feasibility of selecting phage encoded peptide-small molecule hybridstructures was put to the test using the two human antigens plasmakallikrein and cathespin G. A library of phage displaying peptides onthe minor coat protein pill with a complexity of 4.4×10⁹ variants wascreated. The peptides were designed to have two sequences of six randomamino acids flanked by three cysteines (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys (SEQID No. 23); FIG. 3A). An alanine was added to the N-terminus of thepeptide to ensure a correct processing of the signal sequence. AGly-Gly-Ser-Gly (SEQ ID No. 20) linker was placed between the thirdcysteine and the gene-3-protein. As phage with the disulfide-freegene-3-protein had about a 100-fold reduced infectivity compared towild-type phage, large quantities of phage particles were produced. A1-liter culture incubated over night at 30° C. yielded typically10¹¹-10¹² infective particles. About 10¹² purified infective phageparticles were chemically modified with the TBMB scaffold and incubatedwith either of the two biotinylated proteases. Binding phage werecaptured on magnetic streptavidin beads and subjected to two furtherselection rounds. Increasing numbers of phage captured in the second orthird selection round indicated that specific binders were enriched.Sequencing of the peptides revealed various consensus sequences eitherin one or even both of the loops (FIGS. 3B and 3C). The DNA of theselected peptides was amplified by population PCR and inserted into anew plasmid for periplasmic expression of the peptides as D1-D2 fusionprotein. Peptide fusion proteins that either showed sequencesimilarities to other selected clones or that were found in multiplecopies, were expressed, purified, chemically modified and tested fortheir inhibitory activity. The best plasma kallikrein and cathepsin Ginhibitors had an IC₅₀ of 400 nM (PK2 and PK4) and 100 nM (CG2 and CG4)respectively when tested as a D1-D2 fusion.

Example 3 Screening

In this example, affinity maturation of human plasma kallikreininhibitors is described.

The comparison of the amino acid sequences of clones selected againsthuman plasma kallikrein revealed that different groups of clones hadhigh sequence similarity mainly in one of the potential binding loops.We assumed that the bi-cyclic molecules were predominately interactingwith the conserved binding loop while the loop with diverse amino acidcompositions had not evolved for optimal interaction with the protease.Therefore new phage libraries were created with peptides having both, aloop with a sequence of one of the three consensus regions found in theselection with plasma kallikrein and a loop with six random amino acids(FIG. 4A). Phage panning with higher selection pressure using lowerantigen concentrations (1 nM to 200 pM), yielded clones having aconsensus sequence in the second interaction loop (FIG. 4B). Inhibitionassays revealed that the IC₅₀ of the best inhibitor (PK15) was improvedby about a factor 20 (20 nM) when tested as a D1-D2 fusion.

Example 4 Characterisation of Complexes

Activity and specificity of chemically synthesized inhibitors isinvestigated.

Synthetic peptides of the four best human plasma kallikrein inhibitorsisolated in the first selection (PK2, PK4, PK6, PK13) and of the bestinhibitor from the affinity maturation selection (PK15) were produced bysolid phase synthesis. The peptides were designed to have an alaninewith a free amino group at the N-termini and an amidated glycine at theC-termini to represent exactly the charge and chemical environment ofthe phage displayed peptides. The synthetic TBMB reacted peptides werefound to have about 10-fold lower IC₅₀'s than the corresponding TBMBreacted D1-D2 fusion peptides (Table A; FIG. 5). The lower affinity ofthe peptides as D1-D2 fusion may originates from intramolecular bindingof the peptide to the gene-3-protein domains and hence a lower apparentinhibitor concentration. The apparent K_(i) of the TBMB modified peptidePK15 was calculated with the equation of Cheng and Prusoff and was foundto be 1.5 nM (Cheng, Y. and Prusoff, W. H., Biochem. Pharmacol., 1973).The IC₅₀'s of the linear, non-constrained peptides were at least250-fold higher than the ones of the TBMB modified peptides (Table A):

TABLE A  Mass (Da) IC₅₀ (nM) Linear Bi-cyclic Linear Bi-cyclic CloneAmino acid sequence peptide peptide peptide peptide PK2H-ACSDRFRNCPLWSGTCG-NH₂ 1871.2 1985.3 >10′000 28.6 (SEQ ID No. 1) PK4H-ACSTERRYCPIEIFPCG-NH₂ 1942.9 2055.9    7181 33 (SEQ ID No. 2) PK6H-ACAPWRTACYEDLMWCG-NH₂ 1974.8 2088.7    5707 21.2 (SEQ ID No. 3) PK13H-ACGTGEGRCRVNWTPCG-NH₂ 1764.8 1879.1 >10′000 39.1 (SEQ ID No. 4) PK15H-ACSDRFRNCPADEALCG-NH₂ 1825 1939.4 >10′000 1.7 (SEQ ID No. 5)

Mass spectrometric analysis of inhibitor incubated with human plasmakallikrein showed a mass drop of 18 Da suggesting that a peptide bond inone of the loops of the inhibitor was hydrolysed. The inhibitoryactivity (IC₅₀) of kallikrein-treated inhibitor, however, was as good asthe one of the intact, bi-cyclic TBMB modified peptide PK15.

The specificities of the five inhibitors were tested by measuring theinhibitory activity towards mouse plasma kallikrein (79% sequenceidentity) or the homologous human serine proteases factor XIa (sharingthe highest sequence identity with human plasma kallikrein within thehuman serine proteases; 63%) and thrombin (36% sequence identity).Neither the mouse plasma kallikrein nor one of the homologous humanserum proteases were inhibited at the highest concentration tested (10μM).

Example 5 Use of Entities Identified in Methods of the Invention

In this example, inhibition of contact activation in human plasma by ahuman plasma kallikrein inhibitor is demonstrated.

Human plasma kallikrein plays a key role in the first events in contactactivation. The ability of TBMB modified peptide PK15 to inhibit contactactivation was tested by measuring the prolongation of the thrombinactivation time in human plasma in the presence of varying inhibitorconcentrations. Thrombin is the last enzyme in the activation cascade ofthe blood coagulation pathway that is activated. At 50 nM inhibitorconcentration, TBMB modified peptide PK15 delayed thrombin formationwhile aprotinin, a 6 kDa protein inhibitor of human plasma kallikreinhad no effect (FIGS. 8A and 7B). At an inhibitor concentration as highas 5 μM the lag time of thrombin activation was more prolonged byaprotinin than by the small molecule inhibitor. Aprotinin is a broadspectrum inhibitor and may inhibit other proteases in the intrinsicpathway when used at a high concentration. In a different assay, wetested whether TBMB modified peptide PK15 can suppress the activation offactor XIIa and plasma kallikrein in human plasma of three differentdonors. The activation of the two proteases could essentially besuppressed at 5 μM of TBMB modified peptide PK15. We estimate that abouta 30-fold higher concentration of aprotinin is necessary to obtain asame inhibition effect. (FIG. 8C).

Example 6 Structure Determination of TBMB Modified Peptide PK15

The conformation of TBMB modified peptide PK15 was determined by 2D ¹HNMR spectroscopy in aqueous solution at pH 6.6. Chemical shiftassignments were achieved by standard methods. Analysis of the NOESYspectra provided evidence for a defined backbone conformation. Notableare the chemical shifts of the three protons of the central benzene ringthat could be resolved as a result of their different spacialenvironments. Average solution structures were calculated usingNOESY-derived distance restraints (FIG. 6).

Summary of Examples 1 to 6

We have demonstrated the invention with reference to phage displaytechnology to encode the peptide fraction of non-natural small moleculestructures (i.e. complexes according to the present invention). Thegenetic encoding allows the facile generation, selection andamplification of very large combinatorial repertoires. A majordifficulty in this approach was to tether the phage encoded peptiderepertoires to the small molecule core. We developed a convenientsynthesis strategy and established optimal reaction conditions in anumber of experiments. Reagent concentrations, solvent composition andreaction temperature had to be chosen carefully to attach specificallylinear peptides on phage to small molecules while sparing the phageparticles. A specific phage with disulfide-free gene-3-proteins is usedto help prevent the generation of product mixtures through reaction ofthe small molecule with cysteine residues of the phage coat.

We have chosen human plasma kallikrein and cathepsin G as targets totest the efficiency of the in vitro selection techniques of theinvention. Molecules with affinities in the lower nanomolar range wereisolated against both targets and confirmed that the proposed selectionstrategy and the molecule design can yield high affinity binders. Whenassessing the specificity of the human plasma kallikrein inhibitors, wefound that neither the mouse plasma kallikrein nor homologous humanplasma proteases as factor XIa or thrombin were inhibited. This findingwas pleasing since the generation of specific small molecular weightinhibitors to human plasma kallikrein (Young, W. B. et al., Bioorganicand Medicinal Chemistry Letters, 2006) and other human serine proteasesis not trivial (reviewed in Abbenante, G. and Fairlie, D. P., MedicinalChemistry, 2005 and Turk, B., Nature Rev. Drug Discovery, 2006). Theaccess of the small molecule structures to chemical synthesis allows thereplacing of specific amino acids with non-natural building blocks andhence the further improving of the affinity of the inhibitors.

Structure determination of one of the plasma kallikrein inhibitors byNMR in solution suggested that the molecule has a defined backboneconformation. As anticipated, the hydrophobic benzene ring forms thecore of the molecule. However, none of the amino acid side chainsdensely packed with the benzene ring for this particular singlepolypeptide-connector compound combination. Alternative scaffolds withchemical structures that offer more possibilities to interact with thepeptide backbone or amino acid side chains may advantageously be used toobtain a denser packing of the peptide fraction if desired. The hydrogenatoms of the 1,3,5-tris-(bromomethyl)-benzene scaffold at the ringpositions 2, 4 and 6 could for example be replaced by three identicalchemical substituents.

In the selections demonstrated herein, we used a molecule design inwhich a peptide is tethered via three linkages to a small moleculescaffold to obtain a bi-cyclic peptide structure. Of course, thecreation of alternative molecule architectures in which the peptideloops are cleaved by proteases before selection to obtain smallmolecules with discrete peptide moieties may also be used inselection/screening embodiments. In fact, structures with two discretepeptide moieties were generated in this work when the TBMB modifiedinhibitor PK15 was cleaved by human plasma kallikrein upon incubationwith the enzyme. The singly digested molecule was found to have aninhibitory activity that was as good as the non-hydrolized form.Cleavage of the peptide loops also offers the possibility to attachadditional chemical structures to the nascent amino or carboxy terminithrough further chemical reactions.

We have assessed the therapeutic potential of the evolved human plasmakallikrein inhibitor by testing its ability to inhibit contactactivation in human plasma. In cardiac surgery involving cardiopulmonarybypass (CPB) contact of blood with the artificial surface of the CPBmachine and tubing activates multiple plasma protease pathways. Seriouscomplications can result, including the systemic inflammatory responsesyndrome (SIRS), a whole body inflammatory state that can compromiseheart and lung function in patients (Miller, B. E. et al., J. ofCardiothoracic and Vascular Anesthesia, 1997). Plasma kallikrein plays akey role in the first events of contact activation and in theamplification of other protease pathways, such as the fibrinolytic andcomplement systems. A common strategy to suppress contact activationduring cardiac surgery is to block the activity of plasma kallikreinwith aprotinin, a 6 kDa broad spectrum protease inhibitor from bovinelung tissue. The inhibitor binds plasma kallikrein with a K_(i) of 30 nMand hence interrupts the intrinsic coagulation pathway throughsuppression of factor XII activation. Furthermore, inhibition of plasmakallikrein decreases the conversion of plasminogen to plasmin and hencereduces fibrinolylsis and associated bleeding. Aprotinin is also adirect inhibitor of plasmin (K_(i)=3 nM). It is thought that directinhibition of plasmin is the major mechanism of the antifibrinolyticeffects leading to reduction of blood loss and reduced need oftransfusion. The drug has also adverse effects as anaphylaxis and renaltoxicity (reviewed in Mandy A. M. and Webster N. R., Br. J. Anaesth.,2004). An alternative plasma kallikrein inhibitor based on the humankunitz domain scaffold (6 kDa) has recently been developed (Markland, W,et al., Biochemistry, 1996). The drug has a significantly higheraffinity (K_(i)=30 pM) and specificity for plasma kallikrein and isexpected to be less immunogenic due to its human framework. It iscurrently tested in phase 2 clinical trials (Dyax Corp., www.dyax.com).Even though our newly developed lead inhibitor has about a 50-fold loweraffinity for human plasma kallikrein than the product kunitz domainbased inhibitor, it proved to suppress efficiently contact activation exvivo. Its smaller size (2 kDa) allows not only the facile chemicalsynthesis but also advantageously minimises the risk of an immunogenicreaction and makes the compound an attractive lead inhibitor fordevelopment/for use in CPB operations.

Example 7 Non-Covalent Interactions

The connector compound of the invention provides the further advantageof influencing/stabilising or imposing conformational constraints on thetarget polypeptide by virtue of non-covalent bonds formed between theconnector compound and the target polypeptide. These are advantageouslyprovided in addition to the covalent bonds between the connectorcompound and the target polypeptide.

It should be noted that such bonding and constraints are not provided byprior art techniques such as crosslinking. Firstly, crosslinking agentsare typically too small and/or too flexible to contribute conformationalconstraint. Secondly, in the specific example of known crosslinkingdiscussed above (e.g. Roberts US2003/0235852A1) the bivalent linker issmall (propyl) and highly flexible by intentional design and there is noevidence that this produced any non-covalent interactions or imposed anyconformational constraint beyond the joining of two residues within thepolypeptide. In any case, this prior art crosslinker is only bivalent.

In this example we demonstrate that advantageous non-covalent bondingbetween the connector compound and the target polypeptide of theinvention is possible.

The structure of a human plasma kallikrein inhibitor generated with themethod of the invention (see above examples) was solved by NMR. In theproposed structure, several carbon atoms of the polypeptide are in closeproximity (<4 angstrom) to the carbon atoms of the connector compound.This suggests that noncovalent interactions are present, in this examplehydrophobic interactions, between the core and the polypeptide of theinvention.

These interactions are:

Ser3 CB Rng C26 3.62 Å Ser3 CB Rng C2  4.0 Å Ser3 CB Rng CMe2 3.63 ÅCys2 CB Rng CMe2 2.56 Å Cys9 CB Rng C29 3.13 Å Cys9 CB Rng C9 3.32 ÅPro10 CG Rng CMe9  3.8 Å Pro10 CD Rng CMe9 3.13 Å Cys16 CB Rng C16 3.43Å Cys16 CB Rng C26 3.79 Å

In addition, hydrogen-hydrogen interactions between hydrogen atoms ofthe polypeptide and hydrogen atoms of the connector compound weredetected by 1H-NMR NOESY spectroscopy.

Thus, multiple classes of non-covalent interaction between the connectorcompound and target polypeptide of the invention are demonstrated. Theseadvantageously provide further conformational constraint to thepolypeptides of the invention.

Example 8 Phage Encoded Combinatorial Chemical Libraries

Overview

Phage display technology has previously proved effective for makingtherapeutic antibodies from combinatorial libraries but difficult toapply for making small molecule drugs. Here we describe a phage strategyfor the selection of mimics of macrocyclic compounds produced by thenon-ribosomal peptide synthases. The peptide repertoires were designedwith three reactive cysteine residues, each spaced apart by severalrandom amino acid residues, and fused to the phage gene-3-protein.Conjugation with a connector compound (in this exampletris-(bromomethyl)benzene) via the reactive cysteines generatedrepertoires of peptide conjugates with two peptide loops anchored to amesitylene core. Iterative affinity selections yielded several enzymeinhibitors; after further mutagenesis and selection, we isolated a leadinhibitor (PK15)=1.5 nM) specific to human plasma kallikrein thatefficiently interrupted the intrinsic coagulation pathway in humanplasma tested ex vivo. Thus it is demonstrated that this approachprovides a powerful means of generating and screening such macrocyclemimics.

Background

The discovery of novel ligands to receptor, enzyme and nucleic acidtargets represents the first stage in the development of therapeuticdrugs. For drugs based on small organic ligands, high throughputscreening (HTS) has proved a popular strategy; large libraries ofcompounds are synthesized (or purchased) and each compound assayed forbinding to the targets. With the use of robots it is possible to screen10⁵-10⁶ compounds per day, but the hits usually require furtherchemistry to improve their binding affinity and target specificity. Fordrugs based on nucleic acids, peptides or proteins, biological selectionmethods offer an alternative strategy. These methods (such as phagedisplay, ribosome display, mRNA display or RNA/DNA aptamer technologies)rely on (a) creating a diverse library wherein the phenotype (binding totarget) of each member of the library is linked to its genotype (theencoding DNA or RNA), and (b) an iterative cycle in which librarymembers are selected for binding to target, and then amplified (byreplication in a host cell, or by copying of the encoded nucleic acid invitro). At each round of selection the binders are thereby enriched overthe non-binders. Very large libraries (10⁹-10¹³ members) can beefficiently screened by a few rounds of selection and lead hits can berefined by mutation and further selection³. The approach is verypowerful and has been used to create therapeutic antibodies such asHumira™. Several attempts have been made to develop selection methodsfor the isolation of small organic ligands. Typically DNA is used as atag that can be readily synthesized, sequenced, amplified and/orhybridized. For example, small molecules can each be conjugated to aunique DNA⁶ (or bacteriophage⁷) tag, and the conjugates mixed togetherto create a tagged small molecule library. After selection of thelibrary against the target, the small molecule “hits” can be identifiedby the sequences of their (amplified) tags. Alternatively the DNA tagscan be introduced during the synthesis of combinatorial chemicallibraries. For example, small molecules and a corresponding tag aresynthesised in parallel on the same bead⁸, or hybridisation of the tagis used to govern the route of chemical synthesis⁹. From such librariesthe synthetic route (and thereby structure) of the selected hits can bededuced from the sequence of the tag. Notwithstanding their ingenuity,these methods suffer from a common disadvantage; the small molecule islinked to the DNA tag only during the first round of selection,rendering iterative cycles impossible (and limiting application to smalllibraries). Thus the prior art presents numerous difficulties.

In this example, we demonstrate that the invention can be used tochemically modify peptides on phage during the selection process tocreate mimics of peptide macrocyclic compounds. Recently methods havebeen described for tethering peptides through reactive side chains (eg.cysteines) to the functional groups of an organic scaffold¹², andthereby generating polycyclic peptide conjugates comprising an organiccore decorated with peptide loops. As the structures are reminiscent ofthe peptide macrocyclic drugs, we explored the possibility of creatingand selecting libraries of such conjugates on filamentous phage (FIG. 9a). Whereas peptide macrocycles are normally made in vivo bynon-ribosomal peptide synthases, our strategy uses ribosomal synthesisin vivo then chemical conjugation ex vivo.

Results

Conjugation of Organic Scaffold to Peptides Displayed on Phage

We used the small organic compound tris-(bromomethyl)benzene (TBMB) as ascaffold (connector compound) to anchor peptides containing threecysteine residues^(12,15) (FIG. 9a ). The reaction occurs in aqueoussolvents at room temperature, and the three-fold rotational symmetry ofthe TBMB molecule ensures the formation of a unique structural andspatial isomer.

We first elaborated the reaction conditions for conjugation of thepeptide ^(N)GCGSGCGSGCG^(C) (SEQ ID No. 15) fused to the soluble D1-D2domains of the phage pill, analysing the molecular weight of theproducts by mass spectrometry. However, we were unable to selectivelyconjugate the three cysteine residues of the peptide with TBMB whilesparing the disulphide bridges of D1 and D2 (C7-C36, C46-C53,C188-C201). This prompted us to take advantage of a disulfide-freegene-3-protein recently developed by Schmidt F. X. and co-workers¹⁶. Thepeptide-D1-D2 (disulfide free) fusion protein was reduced withtris-(carboxyethyl)phosphine (TCEP), the TCEP removed and TBMB added. Aconcentration of 10 μM TBMB was sufficient for quantitative reactionwith of peptide-fusion protein at 30° C. in one hour, givingpredominantly one product with the expected molecular mass (Δ massexpected=114 Da; FIG. 10a ). No product was detected with the(disulfide-free) D1-D2 protein. Unexpectedly, we found that reaction ofTBMB with peptide-D1-D2 (disulfide-free) fusions containing only twocysteine residues (^(N)AGSGCGSGCG^(C)-(SEQ ID No. 17)D1-D2) yielded aproduct with a molecular mass consistent with reaction of both cysteinesand the α-amino group at the peptide N-terminus (FIGS. 15a and 15b ).Similarly, the reaction of TBMB with a peptide-D1-D2 (disulfide free)fusions having one cysteine and a lysine (^(N)AGSGKGSGCG^(C) (SEQ ID No.18)-D1-D2) yielded a molecular mass consistent with the reaction of thecysteine, the α-amino group of the N-terminus and the ε-amino group ofthe lysine (FIGS. 15c and 15d ). Having identified suitable conditions,we reacted TBMB with (disulfide-free p3) phage bearing the peptide^(N)GCGSGCGSGCG^(C) (SEQ ID No. 15). This led to a small loss (5-fold)of phage infectivity (FIG. 10b ).

Creation of Polycyclic Peptide Library and Affinity Selection

We designed a library of peptides comprising two sequences of six randomamino acids flanked by three cysteines (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys (SEQID No. 23); FIG. 11a ) for display on the (disulphide-free p3) phage. Analanine residue was added to the N-terminus of the peptide to ensure acorrect processing of the signal sequence. A Gly-Gly-Ser-Gly (SEQ ID No.20) linker was placed between the third cysteine and the gene-3-protein.As the (disulfide-free p3) phage had a 100-fold reduced infectivitycompared to wild-type phage, large quantities of phage particles wereproduced from the library (estimated 4.4×10⁹ variants). A 1-literculture incubated over night at 30° C. yielded typically 10¹¹-10¹²infective particles.

We tested the library of polycyclic peptides for binding and inhibitionof the human proteases plasma kallikrein and cathepsin G. About 10¹²purified infective phage particles were chemically modified with TBMBand then incubated with the biotinylated target proteins. After captureon magnetic streptavidin or avidin beads, the enriched phage weretreated to two further rounds of selection, each round comprisingamplification (by bacterial infection), chemical conjugation and capturewith the biotinylated targets. The phage titre increased after thesecond and third rounds suggesting enrichment of specific binders. DNAencoding the peptides was PCR-amplified from the selected population ofphage in the third round, and recloned for periplasmic expression aspeptide-D1-D2 (disulfide free D1-D2) fusion proteins and sequenced. Thisrevealed consensus sequences in one or both of the peptide loops (FIGS.11b and 11c ) and several were expressed, purified, conjugated with TBMBand tested for their inhibitory activity to protease. The best plasmakallikrein and cathepsin G inhibitors had an IC₅₀ of 400 nM (PK2 andPK4) and 100 nM (CG2 and CG4) respectively when tested as a D1-D2fusion. Since we screened the phage selected clones for inhibition(rather than binding) we can not state whether also molecules wereselected that bind to the proteases but do not inhibit them. However,the finding that the vast majority of clones tested after the phageselection displayed inhibitory activities suggests that predominantlyinhibitors were selected.

Affinity Maturation of Human Plasma Kallikrein Inhibitors

Most of the sequences of the kallikrein binders revealed consensussequences in one or other of the peptide loops. Three new libraries werecreated with one of the three consensus regions in one loop and sixrandom amino acids in the other loop (FIG. 12a ). The libraries weremixed and phage panned under stringent conditions (1 nM to 200 pMbiotinylated kallikrein). The random sequence converged to a newconsensus, yielding clones with consensus sequences in both loops (FIG.12b ). Inhibition assays revealed that the IC₅₀ of the best inhibitor(PK15) was 20 nM when tested as a D1-D2 fusion.

Activity and Specificity of Chemically Synthesized Inhibitors

Synthetic peptides corresponding to four kallikrein inhibitors from theprimary selection (PK2, PK4, PK6 and PK13) and the best inhibitor fromthe affinity maturation selection (PK15) were produced by solid phasechemical synthesis. The peptides had an alanine residue at theN-terminus and an amidated glycine at the C-terminus to represent thecharge and chemical environment of the phage displayed peptides. TheTBMB conjugated synthetic peptides were at least 250-fold more potentinhibitors of kallikrein activity than the unconjugated peptides (Table1).

TABLE 1  Chemically synthesized peptide inhibitors. Indicated are themolecular masses and the inhibitory activities before and after themodification of the peptides with TBMB: Mass (Da) IC₅₀ (nM) LinearBi-cyclic Linear Bi-cyclic Clone Amino acid sequence peptide peptidepeptide peptide PK2 H-ACSDRFRNCPLWSGTCG-NH₂ 1871.2 1985.3 >10′000 28.6(SEQ ID No. 1) PK4 H-ACSTERRYCPIEIFPCG-NH₂ 1942.9 2055.9    7181 33(SEQ ID No. 2) PK6 H-ACAPWRTACYEDLMWCG-NH₂ 1974.8 2088.7    5707 21.2(SEQ ID No. 3) PK13 H-ACGTGEGRCRVNWTPCG-NH₂ 1764.8 1879.1 >10′000 39.1(SEQ ID No. 4) PK15 H-ACSDRFRNCPADEALCG-NH₂ 1825 1939.4 >10′000 1.7(SEQ ID No. 5) The amino acid sequences of five plasma kallikreininhibitors (17-mers) are shown. The sequences of the synthetic peptidesderive from the clones PK2, PK4, PK6, PK13 (isolated in phage selectionsusing library 1) and from clone PK15 (an affinity matured clone isolatedfrom library 2).

They were more potent inhibitors than the peptide-D1-D2 conjugates by afactor of more than ten (Table 1; FIG. 13); presumably this is due tobinding of the conjugated peptide moiety to the D1-D2 moiety. Theapparent inhibition constant (K_(i)) of the peptide conjugate PK15 (FIG.9b ) was calculated to be 1.5 nM using the equation of Cheng andPrusoff¹⁷. Incubation of the conjugate PK15 with kallikrein leads tohydrolysis of a peptide bond after prolonged incubation (90% cleavageafter 24 h at 37° C.), as shown by a mass gain of 18 Da, but theinhibitory activities of cleaved and uncleaved samples proved similar(IC₅₀ 2.2 nM and 1.6 nM respectively).

The five inhibitors were also tested against mouse plasma kallikrein(79% sequence identity) or the homologous human serine proteases factorXIa (63% sequence identity) and thrombin (36% sequence identity). Noneinhibited these enzymes at the highest concentration tested (10 μM).

Interruption of the Intrinsic Coagulation Pathway by a Human PlasmaKallikrein Inhibitor

Human plasma kallikrein plays a key role in the first events of theintrinsic coagulation pathway by converting factor XII to factor XIIawhich then acts on the next protease in the pathway. We tested whetherconjugate PK15 could inhibit the activation of factor XIIa in humanplasma samples. The pathway was triggered with caolin and the activityof factor XIIa was measured with a colorimetric substrate. The activityof XIIa was halved in the presence of 160 nM conjugate PK15 (FIG. 16).By comparison 5 μM of aprotinin, a 6 kDa bovine serine proteaseinhibitor also used clinically as a plasma kallikrein inhibitor(K_(i)=30 nM), was required for the same effect.

Structure Determination of TBMB Modified Peptide PK15

2D ¹H NMR spectra of the conjugate PK15 were recorded and a sequencespecific alignment of the chemical shifts of the TOCSY and NOESY spectrawas possible. A conformation of the inhibitor calculated on theNOESY-derived distance restraints is shown in FIG. 14. The two peptideloops are arranged around the mesitylene core to which they arecovalently attached but do not interact with each other. The loops donot pack densely against the core but several carbon atoms of thepolypeptide (Cys 9 CB, Cys16 CA, Gly 17 CA) are within 4 Å of atoms ofthe molecular core suggesting there may be some hydrophobicinteractions.

Discussion of Example 8

We have shown how the reaction of tris-(bromomethyl)benzene (TBMB)¹²with libraries of cysteine-rich peptides displayed on filamentousbacteriophage generates conjugates (complexes according to the presentinvention) amenable to iterative selection. It was a challenge toconjugate the displayed peptide while sparing the phage, and we had tovary reagent concentrations, solvent composition and reactiontemperature, and also use phage lacking disulfides in thegene-3-protein. From a library of >10⁹ members and iterative selectionswe succeeded in isolating potent human plasma kallikrein inhibitors(<2000 Da). Our lead inhibitor (PK15) with K_(i)=1.5 nM efficientlyinterrupted the intrinsic coagulation pathway in human plasma tested exvivo, and was highly specific: it did not inhibit mouse plasmakallikrein or the homologous human plasma proteases factor XIa andthrombin.

Our repertoire was built from 17 residue peptides with three cysteines,each spaced apart by six random amino acids. After conjugation with TBMBthe peptides are expected to form two six-residue loops attached to amesitylene core, as indeed confirmed by the structure of the PK15kallikrein inhibitor solved by NMR (FIG. 14). Such polycyclic peptidesshould have advantages over both disulfide-bonded and linear peptides.The advantages of polycyclic peptides over disulfide bonded peptides arethat the covalent carbon-sulfur bonds once formed are inert toexchange¹⁸, and are also stable in reducing environments¹⁸. Theadvantage of polycyclic peptides over linear peptides is that they arecross-linked and more constrained. This has two main consequences: (a)constrained peptides are expected to bind more tightly to targets (dueto the smaller loss of conformational entropy). Our literature review ofpeptide inhibitors isolated by phage display shows that the majoritycontain disulphides, and have inhibition constants in the micromolarrange (Table 3).

TABLE 3  Phage selected peptide inhibitors. Target Peptide sequenceAffinity Reference Prostate specific CVAYCIEHHCWTC K_(D) = 2.9 μM 1(SEQ ID antigen (PSA) No. 24) Human kallikrein 2 SRFKVWWAAF IC₅₀ =3.4 μM 2 (SEQ ID No. 25) Urokinase-type CSWRGLENHRMC K_(i) = 6.7 μM 3(SEQ ID plasminogen No. 26) activator (uPA) Urokinase-type CPAYSRYLDCK_(i) = 0.4 μM 4 (SEQ ID plasminogen No. 27) activator (uPA)Chymotrypsin CCFSWRCRC K_(i) = 1 μM 5 (SEQ ID No. 28 TF-fVIIEEWEVLCWTWETCER IC₅₀ = 1.5 nM 6 (SEQ ID No. 29) AngiotensinGDYSHCSPLRYYPWW K_(i) = 2.8 nM 7 (SEQ ID converting KCTYPDP No. 30)enzyme 2 (ACE2) ErbB-2 KCCYSL K_(i) = 30 μM 8 (SEQ ID No. 31) UreaseYDFYWW IC₅₀ = 30 μM 9 (SEQ ID No. 32) Pancreatic CQPHPGQTC IC₅₀ = 16 μM10 (SEQ ID lipase No. 33) Beta-lactamase CVHSPNREC IC₅₀ = 9 μM 11(SEQ ID No. 34 DNase II CLRLLQWFLWAC K_(i) = 0.2 μM 12 (SEQ ID No. 35)Indicated are the sequences of the peptides, the enzyme targets and thebinding affinities. The cysteine residues that form disulfide bridgesare underlined.

Only two peptide inhibitors were as potent as PK15; both contained adisulfide bond and at least two tryptophan residues. This suggests thatthe constrained conformation and the possibility of hydrophobicinteractions are key for these high affinities; (b) constrained (andcross-linked) peptides should also be more resistant to cleavage and/orinactivation than linear peptides. Indeed in our work the inhibitor PK15was cleaved in one of the loops after prolonged incubation with humanplasma kallikrein, but remained intact and active.

The polycyclic conjugates are amenable to both genetic and chemicalengineering. The molecular weight of PK15 (1939.4 Da) is higher thanseveral peptide macrocyclic drugs (Table 4), but it would be possible touse shorter loops. For example by altering the spacing of the cysteines,the loop length is readily varied, or even extra segments added to thepeptide termini.

TABLE 4 Size comparison of macrocyclic drugs. Molecular mass Name Cyclesize(s) (Da) Application Actionmycin 16, 16 1255.42 anticancerAmphotericin B 38 924.08 antifungal Azithromycin 15 748.88 antibioticCaspofungin 21 1093.31 antifungal Cyclosporin 32 1202.61immunosupression Daptomycin 31 1619.71 antibiotic Erythromycin 14 733.93antibiotic Ixabepilone 16 506.70 anticancer Ocreotide 20 1019.24 hormoneOxytoxin 20 1007.19 hormone Polymyxin B 23 1301.56 antibiotic Rapamyzin29 914.17 immunosupression Rifabutin 27 847.01 antibiotic Vancomycin 16,16, 12 1449.30 antibiotic

Further variations could include mutagenesis of the loops (as with theaffinity maturation of PK15); proteolytic cleavage in one or both loopsto generate peptide segments “branched” at the cysteines; chemicalconjugation to the nascent peptide termini after loop cleavage²¹; or theuse of variant organic cores. For example, a larger organic core, or onewith more functional groups could interact more extensively with theloops or with the target, and could also be used to introduce entirelynew functions such as fluorescence. If these operations were performedon the phage-displayed conjugate, the variations would be selectable byan iterative process. As the peptide conjugates are also amenable tochemical synthesis, further variations (such as the substitution byunnatural amino acids) could be introduced synthetically.

Inhibitors of human plasma kallikrein are being developed clinically fortreatment of hereditary angiodema and coronary bypass surgery, but ithas proved difficult to make small molecules that are specific for thekallikrein (reviewed in 22,23). The fact that we so readily obtained ahigh affinity and highly specific inhibitor by iterative selection ofpolycyclic peptide conjugates on phage augers well for this strategy.

Materials and Methods

Chemical Modification of Peptide Repertoires with TBMB on Phage

Phage peptide libraries that are based on the plasmid fdg3p0ss2116 werecloned and produced as described below. Typically 1011-1012 t.u. of PEGpurified phage were reduced in 20 ml of 20 mM NH4HCO3, pH 8 with 1 mMTCEP at 42° C. for 1 hr. The phage were spun at 4000 rpm in avivaspin-20 filter (MWCO of 10,000) to reduce the volume of thereduction buffer to 1 ml and washed twice with 10 ml ice cold reactionbuffer (20 mM NH4HCO3, 5 mM EDTA, pH 8). The volume of the reduced phagewas adjusted to 32 ml with reaction buffer and 8 ml of 50 M TBMB in ACNwere added to obtain a final TBMB concentration of 10 μM. The reactionwas incubated at 30° C. for 1 hr before non-reacted TBMB was removed byprecipitation of the phage with ⅕ volume of 20% PEG, 2.5 M NaCl on iceand centrifugation at 4000 rpm for 30 minutes.

Phage Selections with Human Plasma Kallikrein and Cathepsin G

Biotinylated human plasma kallikrein and cathepsin G (5 to 20 μg; theprotocol used for the biotinylation can be found below) were blocked byincubation in 0.5 ml washing buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl,10 mM MgCl₂, 1 mM CaCl₂) containing 1% BSA and 0.1% tween 20 for 30minutes. The chemically modified phage (typically 10¹⁰-10¹¹ t.u.dissolved in 2 ml washing buffer) were blocked by addition of 1 ml ofwashing buffer containing 3% BSA and 0.3% tween 20 and incubation for 30minutes. 3 ml blocked phage were pipetted to 0.5 ml blocked antigen andincubated for 30 minutes on a rotating wheel at RT. 50 μl magneticsteptavidin beads (Dynal, M-280 from Invitrogen, Paisley, UK) wereblocked by incubation in 0.5 ml of washing buffer containing 1% BSA and0.1% tween 20 for 30 minutes. The blocked beads were added to thephage/antigen mixture and incubated for 5 minutes at RT on a rotatingwheel. The beads were washed 8 times with washing buffer containing 0.1%tween 20 and twice with washing buffer before incubation with 100 μl of50 μM glycine, pH 2.2 for 5 minutes. Eluted phage were transferred to 50μl of 1 M Tris-Cl, pH 8 for neutralization, incubated with 50 ml TG1cells at OD₆₀₀=0.4 for 90 minutes at 37° C. and the cells were plated onlarge 2YT/chloramphenicol plates. Two additional rounds of panning wereperformed using the same procedures. In the second round of selection,neutravidin-coated magnetic beads were used to prevent the enrichment ofstreptavidin-specific peptides. The neutravidin beads were prepared byreacting 0.8 mg neutravidin (Pierce, Rockford, Ill., USA) with 0.5 mltosyl-activated magnetic beads (Dynal, M-280 from Invitrogen, Paisley,UK) according to the supplier's instructions.

Screening Selected Clones for Inhibitory Activity

The genes that encode the peptides selected in the second and thirdround of biopanning were cloned into a pUC119 based vector forexpression of the peptide-D1-D2 fusion proteins (disulfide-free D1-D2protein; the cloning and expression procedures are described below).Oxidized sulfhydryl groups of the peptides were reduced by incubation ofthe protein (1-10 μM) with 1 mM TCEP in 20 mM NH₄HCO₃, pH 8 at 42° C.for 1 hr. The reducing agent was removed by size exclusionchromatography with a PD-10 column (Amersham Pharmacia, Uppsala, Sweden)using 20 mM NH₄HCO₃, 5 mM EDTA, pH 8 buffer. The thiol groups of theproteins were reacted by incubation with 10 μM TBMB in reaction buffer(20 mM NH₄HCO₃, 5 mM EDTA, pH 8, 20% ACN) at 30° C. for 1 hr. Forremoval of non-reacted TBMB and concentration the protein was filteredwith a microcon YM-30 (Millipore, Bedford, Mass.). The concentrations ofthe products were determined by measuring the optical absorption at 280nm. The IC₅₀ was measured by incubating various concentrations of themodified peptide fusion proteins (2-fold dilutions) with human plasmakallikrein (0.1 nM) or cathepsin G (20 nM) and determining the residualactivity in 10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM CaCl₂,0.1% BSA, 0.01% triton-X100. Human plasma kallikrein activity wasmeasured with the fluorogenic substrate Z-Phe-Arg-AMC (Bachem,Bubendorf, Switzerland) at a concentration of 100 μM on a SpectramaxGemini fluorescence plate reader (excitation at 355 nm, emissionrecording at 460 nm; Molecular Devices, Sunnyvale, Calif., USA). Humancathepsin G activity was measured with the colorimetric substrateN-Suc-Ala-Ala-Phe-Pro-pNA (Bachem, Bubendorf, Switzerland) at aconcentration of 1 mM with a Spectramax absorption plate reader(recording at 410 nm; Molecular Devices, Sunnyvale, Calif., USA).

Chemical Synthesis of Bicyclic Peptides

Peptides with a free amine at the N-terminus and an amide at theC-terminus were chemically synthesized on a 25 mg scale by solid phasechemistry (JPT Peptide Technologies, Berlin, Germany). The crudepeptides in 1 ml 70% NH₄HCO₃, pH 8 and 30% ACN (1 mM) were reacted withTBMB (1.2 mM) for 1 hr at RT. The reaction product was purified byreversed-phase high-performance liquid chromatography (HPLC) using a C18column and gradient elution with a mobile phase composed of ACN and 0.1%aqueous trifluoroacetic acid (TFA) solution at a flow rate of 2 ml/min.The purified peptides were freeze-dried and dissolved in DMSO or abuffer of 50 mM Tris-Cl pH 7.8, 150 mM NaCl for activity measurements.

Cloning and Expression of Peptide-D12 Fusion Proteins

The domains D1-D2 of the g3p (comprising amino acid residues 2 to 217 ofthe mature fdg3p) with and without the N-terminally fused peptide^(N)ACGSGCGSGCG^(C) (SEQ ID No. 16) were expressed in E. coli. ThepUC119 based expression vector with a leader sequence and the D1-D2 genewith a C-terminal hexa-histidine tag (here termed pUC119H6D12) waskindly provided by Phil Holliger from the Laboratory of MolecularBiology (LMB) in Cambridge. A plasmid for expression of D1-D2 with theN-terminal peptide was cloned by PCR amplification of the D1-D2 genewith the primers pepd12ba (encoding the peptide sequence) and d12fo andligation into the SfiI/NotI digested pUC119H6D12 vector. The gene forthe expression of disulfide-free D1-D2 with a total of 20 amino acidswas PCR amplified from the vector fdg3p0ss21 with either the primer paird120ssba/d120ssfo, pepd120ssba/d120ssfo, P2cd120ssba/d120ssfo orP1cd120ssba/d120ssfo and SfiI/NotI ligated into pUC119H6D12 forexpression of disulfide-free D1-D2 with and without the N-terminal fusedpeptides ^(N)ACGSGCGSGCG^(C) (SEQ ID No. 16), ^(N)AGSGCGSGCG^(C) (SEQ IDNo. 17) or ^(N)AGSGKGSGCG^(C) (SEQ ID No. 18). All 6 proteins wereexpressed in TG1 E. coli cells at 30° C. for 8 hours and the periplasmicfraction was purified stepwise by Ni-affinity chromatography and gelfiltration on a Superdex 75 column in 20 mM NH₄HCO₃ pH 7.4.

Mass Spectrometric Analysis of Peptide-D12 Fusion Proteins

The molecular masses of the proteins (5-20 μM) before and aftermodification with TBMB were determined by denaturing the proteins in 4volumes of 50% MeOH, 1% formic acid and analysis on a time of flightmass spectrometer with electrospray ionization (Micromass, Milford,Mass., USA). Molecular masses were obtained by deconvoluting multiplycharged protein mass spectra using MassLynx version 4.1.

Creation of the Phage Peptide Library 1

The genes encoding a semi-random peptide with the sequenceAla-Cys-(Xaa)₆-Cys-(Xaa)₆-Cys (SEQ ID No. 19), the linkerGly-Gly-Ser-Gly (SEQ ID No. 20) and the two disulfide-free domains D1and D2 were cloned in the correct orientation into the phage vectorfd0D12 to obtain phage library 1. The vector fd0D12, lacking the genesof the D1 and D2 domains of gene 3 and having a second SfiI restrictionsite was previously created by whole-plasmid PCR amplification offdg3p0ss21 using the primer ecoG3pNba and pelbsfiecofo. The genesencoding the peptide repertoire and the two gene 3 domains werestep-wise created in two consecutive PCR reactions. First, the genes ofD1 and D2 were PCR amplified with the two primer preper and sfi2fo usingthe vector fdg3p0ss21 as a template. Second, the DNA encoding the randompeptides was appended in a PCR reaction using the primer sficx6ba andsfi2fo. The ligation of 33 and 9 μg of SfiI digested fd0D12 plasmid andPCR product yielded 4.4×10⁹ colonies on 12 20×20 cm chloramphenicol (30μg/ml) 2YT plates. Colonies were scraped off the plates with 2YT media,supplemented with 15% glycerol and stored at −80° C. Glycerol stockswere diluted to OD₆₀₀=0.1 in 1 liter 2YT/chloramphenicol (30 μg/ml)cultures and phage were expressed at 30° C. over night (12 to 16 hrs).

Biotinylation of Antigens

Human plasma kallikrein (activated with factor XIIa) was purchased fromInnovative Research (Southfield, Mich., USA) and biotinylated at aconcentration of 1.2 μM with a 5-fold molar excess ofSulfo-NHS-LC-biotin (Pierce, Rockford, Ill., USA) in PBS, pH 7.4/5% DMSOat RT for 1 hr. The biotinylated protein was purified on a PD-10 columnusing a buffer of 50 mM NaAc, pH 5.5, 200 mM NaCl. Readily biotinylatedhuman cathepsin G was purchased from Lee Biosolutions (St. Louis, Mich.,USA).

Subcloning and Expression Screening of Phage Selected Clones

The plasmid DNA of clones selected after the second and third round ofbiopanning was PCR-amplified in a single tube with the primer 21seqbaand flagfo and cloned into the vector pUC119H6D12 at the SfiI and NotIsites for the periplasmic expression of the peptides fused to thedisulfide-free D1 and D2 domains with a C-terminal FLAG tag and ahexa-histidine-tag. The ligated plasmids were electroporated into TG1cells and plated on 2YT/ampicillin (100 μg/ml) plates. Clones expressingthe recombinant protein were identified as follows: Media (2YT with 100μg/ml ampicillin) in 96-deep well plates (1 ml/well) was inoculated withcells of individual colonies and incubated at 37° C. Protein expressionwas induced with 1 mM IPTG when the cultures were turbid and the plateswere shaken 300 rpm at 30° C. o/n. The cells were pelleted bycentrifugation at 3500 rpm for 30 minutes, lysed with washing buffercontaining 1 mg/ml lysozyme and spun at 3500 rpm to pellet the celldebris. The supernatants were transferred to 96-well polysorp plates(Nunc, Roskilde, Denmark) for non-specific adsorbtion. The wells wererinsed twice with washing buffer containing 0.1% tween 20 and blockedwith washing buffer containing 1% BSA and 0.1% tween 20 for 1 hr.Anti-FLAG M2-peroxidase conjugate (Sigma-Aldrich, St. Louis, Mo., USA)was 1:5000 diluted and blocked in washing buffer containing 1% BSA and0.1% tween 20 and added to the plates for 1 hr. The wells were washed (5times with washing buffer containing 0.1% tween 20 and once withoutdetergent) and the bound peroxidase was detected with TMB substratesolution (eBiosciences, San Diego, USA). The plasmid DNA of proteinexpressing clones was sequenced (Geneservice, Cambridge, UK).

Affinity Maturation of Human Plasma Kallikrein Inhibitors

Three peptide phage libraries were created essentially as the library 1(see above) but using the degenerate primer sficx6abc (library 2),sficx6abb (library 3) and sficx6aba (library 4) instead of sficx6ba.Electroporation of the ligation reactions into TG1 cells yielded 9.5×10⁸(library 2), 1.1×10⁹ (library 3) and 1.2×10⁹ (library 4) transformants.Phage of each library were produced in 1 L cultures, purified, pooledand reacted with TBMB. Three rounds of panning were performedessentially as in the selections described in the materials and methodssection but using the biotinylated human plasma kallikrein at a lowerconcentration (1 nM in the 1^(st) and 2^(nd) round, 200 pM in the 3^(rd)round).

Activity and Specificity Measurement of Human Plasma KallikreinInhibitors

Inhibitory activities (IC₅₀) were determined by measuring residualactivities of the enzyme upon incubation (30 min, RT) with differentconcentrations of inhibitor (typically ranging from 10 μM to 0.5 nM).The activities of human plasma kallikrein (0.1 nM) and factor XIa (0.8nM; Innovative Research, Southfield, Mich., USA) were measured withZ-Phe-Arg-AMC (100 μM) and the activity of human thrombin (2 nM;Innovative Research, Southfield, Mich., USA) with Boc-Phe-Ser-Arg-AMC(100 μM) in 10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM CaCl₂,0.1% BSA, 0.01% triton X-100 and 5% DMSO. Recombinant mouse plasmakallikrein from R&D Systems (Minneapolis, Minn., USA) with a signalpeptide was proteolytically activated with 0.5 mg/ml thermolysin at 37°C. for 1 hr. The activity of mouse plasma kallikrein (3 nM) was measuredwith Z-Phe-Arg (SEQ ID No. 36)-AMC (100 μM) in 50 mM Tris-Cl pH 7.5, 10mM CaCl₂ and 250 mM NaCl, 0.1% BSA, 0.01% triton X-100 and 5% DMSO.Inhibitor hydrolysed in one binding loop was generated by incubation ofTBMB modified peptide PK15 with human plasma kallikrein at a molar ratioof 5:1 for 24 hours at 37° C. and subsequent heat inactivation of theenzyme at 60° C. (30 min). Apparent K_(i) values were calculatedaccording to the Cheng and Prusoff equation.

Measurement of Factor XII Activation in Human Plasma

Normal human plasma from single donors was purchased from 3H Biomedical(Uppsala, Sweden). The plasma was centrifuged at 1500×g at 20° C. for 15minutes to obtain platelet-poor plasma (PPP). Aliquots of the PPP werestored in polypropylene tubes at −80° C. Samples of 60 μl PPP containing5, 50, 500 or 5000 nM of aprotinin (Roche, Mannheim, Germany) or TBMBmodified peptide PK15 were prepared. Activation of factor XIIa wasmeasured as follows. 2 μg of kaolin was added to the plasma samples,mixed well and incubated for 20 minutes at 37° C. The samples werediluted 250-fold in 50 mM Tris-Cl pH 7.8, 150 mM NaCl. Plasmakallikrein-like activity was measured with the chromogenic substrateH-D-Pro-Phe-Arg (SEQ ID No. 37)-pNA (100 μM; Bachem, Bubendorf,Switzerland) using an absorption plate reader (absorption at 450 nm;Molecular Devices, Sunnyvale, Calif., USA). The same chromogenicsubstrate is also recognized and modified by plasma kallikrein. However,at the inhibitor concentrations required to reduce the factor XIIaactivity by 50% (160 nM for the TBMB modified peptide PK15 and 5 μM foraprotinin), the plasma kallikrein is essentially inhibited and can notbe measured with the substrate.

Structure Determination of TBMB Modified Peptide PK15

1 mg of TBMB modified peptide PK15 was dissolved in 550 μl 10 mMdeuterated Tris-Cl pH 6.6, 150 mM NaCl, 10 mM MgCl₂, 1 mM CaCl₂ toobtain an inhibitor concentration of 1 mM. Spectra of the inhibitor wererecorded at 800 MHz (Bruker Avance with TCI cryoprobe). Spectralassignments were based on TOCSY and NOESY spectra. There were a total of199 NOE restraints, 77 of which were inter-residue, and 122intra-residue. The structure shown in FIG. 6 is the average structure of50 calculated structure conformers. The program PyMOL was used forstructure analysis and visualization of the molecular models.

REFERENCES FOR MATERIALS AND METHODS SECTION

-   1. Wu, P., Leinonen, J., Koivunen, E., Lankinen, H. & Stenman, U. H.    Identification of novel prostate-specific antigen-binding peptides    modulating its enzyme activity. Eur J Biochem 267, 6212-20 (2000).-   2. Hekim, C. et al. Novel peptide inhibitors of human kallikrein 2.    J Biol Chem 281, 12555-60 (2006).-   3. Hansen, M. et al. A urokinase-type plasminogen    activator-inhibiting cyclic peptide with an unusual P2 residue and    an extended protease binding surface demonstrates new modalities for    enzyme inhibition. J Biol Chem 280, 38424-37 (2005).-   4. Andersen, L. M., Wind, T., Hansen, H. D. & Andreasen, P. A. A    cyclic peptidylic inhibitor of murine urokinase-type plasminogen    activator: changing species specificity by substitution of a single    residue. Biochem J 412, 447-57 (2008).-   5. Krook, M., Lindbladh, C., Eriksen, J. A. & Mosbach, K. Selection    of a cyclic nonapeptide inhibitor to alpha-chymotrypsin using a    phage display peptide library. Mol Divers 3, 149-59 (1997).-   6. Dennis, M. S. et al. Peptide exosite inhibitors of factor Vila as    anticoagulants. Nature 404, 465-70 (2000).-   7. Huang, L. et al. Novel peptide inhibitors of    angiotensin-converting enzyme 2. J Biol Chem 278, 15532-40 (2003).-   8. Karasseva, N. G., Glinsky, V. V., Chen, N. X., Komatireddy, R. &    Quinn, T. P. Identification and characterization of peptides that    bind human ErbB-2 selected from a bacteriophage display library. J    Protein Chem 21, 287-96 (2002).-   9. Houimel, M., Mach, J. P., Corthesy-Theulaz, I., Corthesy, B. &    Fisch, I. New inhibitors of Helicobacter pylori urease holoenzyme    selected from phage-displayed peptide libraries. Eur J Biochem 262,    774-80 (1999).-   10. Lunder, M., Bratkovic, T., Kreft, S. & Strukelj, B. Peptide    inhibitor of pancreatic lipase selected by phage display using    different elution strategies. J Lipid Res 46, 1512-6 (2005).-   11. Sanschagrin, F. & Levesque, R. C. A specific peptide inhibitor    of the class B metallo-beta-lactamase L-1 from Stenotrophomonas    maltophilia identified using phage display. J Antimicrob Chemother    55, 252-5 (2005).-   12. Sperinde, J. J., Choi, S. J. & Szoka, F. C., Jr. Phage display    selection of a peptide DNase II inhibitor that enhances gene    delivery. J Gene Med 3, 101-8 (2001).

REFERENCES TO EXAMPLE 8

-   1. Hüser, J. High-Throughput Screening in Drug Discovery (eds.    Mannhold, R., Kubinyi, H. & Folkers, G.) (Wiley-VCH, Weinheim,    2006).-   2. Bleicher, K. H., Bohm, H. J., Muller, K. & Alanine, A. I. Hit and    lead generation: beyond high-throughput screening. Nat Rev Drug    Discov 2, 369-78 (2003).-   3. Marks, J. D., Hoogenboom, H. R., Griffiths, A. D. & Winter, G.    Molecular evolution of proteins on filamentous phage. Mimicking the    strategy of the immune system. J Biol Chem 267, 16007-10 (1992).-   4. Jespers, L. S., Roberts, A., Mahler, S. M., Winter, G. &    Hoogenboom, H. R. Guiding the selection of human antibodies from    phage display repertoires to a single epitope of an antigen.    Biotechnology (N Y) 12, 899-903 (1994).-   5. Hudson, P. J. & Souriau, C. Engineered antibodies. Nat Med 9,    129-34 (2003).-   6. Doyon, J. B., Snyder, T. M. & Liu, D. R. Highly sensitive in    vitro selections for DNA-linked synthetic small molecules with    protein binding affinity and specificity. J Am Chem Soc 125, 12372-3    (2003).-   7. Woiwode, T. F. et al. Synthetic compound libraries displayed on    the surface of encoded bacteriophage. Chem Biol 10, 847-58 (2003).-   8. Brenner, S. & Lerner, R. A. Encoded combinatorial chemistry. Proc    Natl Acad Sci USA 89, 5381-3 (1992).-   9. Halpin, D. R. & Harbury, P. B. DNA display II. Genetic    manipulation of combinatorial chemistry libraries for small-molecule    evolution. PLoS Biol 2, E174 (2004).-   10. Jespers, L., Bonnert, T. P. & Winter, G. Selection of optical    biosensors from chemisynthetic antibody libraries. Protein Eng Des    Sel 17, 709-13 (2004).-   11. Jespers, L. S. A., Winter, G. P., Bonnert, T. P. & Simon, T. M.    (PCT/GB94/01422).-   12. Timmerman, P., Beld, J., Puijk, W. C. & Meloen, R. H. Rapid and    quantitative cyclization of multiple peptide loops onto synthetic    scaffolds for structural mimicry of protein surfaces. Chembiochem 6,    821-4 (2005).-   13. Driggers, E. M., Hale, S. P., Lee, J. & Terrett, N. K. The    exploration of macrocycles for drug discovery—an underexploited    structural class. Nat Rev Drug Discov 7, 608-24 (2008).-   14. Wessjohann, L. A., Ruijter, E., Garcia-Rivera, D. & Brandt, W.    What can a chemist learn from nature's macrocycles?—a brief,    conceptual view. Mol Divers 9, 171-86 (2005).-   15. Kemp, D. S. & McNamara, P. E. Conformationally restricted cyclic    nonapeptides derived from L-cysteine and    LL-3-amino-2-piperidino-6-carboxylic acid (LL-acp), a potent    b-turn-inducing dipeptide analogue. Journal of Organic Chemistry 50,    5834-5838 (1985).-   16. Kather, I., Bippes, C. A. & Schmid, F. X. A stable    disulfide-free gene-3-protein of phage fd generated by in vitro    evolution. J Mol Biol 354, 666-78 (2005).-   17. Cheng, Y. & Prusoff, W. H. Relationship between the inhibition    constant (K1) and the concentration of inhibitor which causes 50    percent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol    22, 3099-108 (1973).-   18. Cremlyn, R. J. An introduction to organosulfur chemistry (Wiley,    1996).-   19. Huang, L. et al. Novel peptide inhibitors of    angiotensin-converting enzyme 2. J Biol Chem 278, 15532-40 (2003).-   20. Dennis, M. S. et al. Peptide exosite inhibitors of factor Vila    as anticoagulants. Nature 404, 465-70 (2000).-   21. Jackson, D. Y. et al. A designed peptide ligase for total    synthesis of ribonuclease A with unnatural catalytic residues.    Science 266, 243-7 (1994).-   22. Abbenante, G. & Fairlie, D. P. Protease inhibitors in the    clinic. Med Chem 1, 71-104 (2005).-   23. Turk, B. Targeting proteases: successes, failures and future    prospects. Nat Rev Drug Discov 5, 785-99 (2006).-   24. Melkko, S., Scheuermann, J., Dumelin, C. E. & Neri, D. Encoded    self-assembling chemical libraries. Nat Biotechnol 22, 568-74    (2004).-   25. Li, S. & Roberts, R. W. A novel strategy for in vitro selection    of peptide-drug conjugates. Chem Biol 10, 233-9 (2003).-   26. Millward, S. W., Takahashi, T. T. & Roberts, R. W. A general    route for post-translational cyclization of mRNA display libraries.    J Am Chem Soc 127, 14142-3 (2005).-   27. Millward, S. W., Fiacco, S., Austin, R. J. & Roberts, R. W.    Design of cyclic peptides that bind protein surfaces with    antibody-like affinity. ACS Chem Biol 2, 625-34 (2007).

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed aspects and embodiments of the present invention will beapparent to those skilled in the art without departing from the scope ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are apparent tothose skilled in the art are intended to be within the scope of thefollowing claims.

The invention claimed is:
 1. A genetically encoded polypeptide libraryof complexes comprising: (a) a nucleic acid genetically encoding apolypeptide of the polypeptide library; (b) the genetically encodedpolypeptide library displayed on a phage display system, wherein thepolypeptide comprises the sequence (X)IC(X)mC(X)nC(X)o (SEQ ID No. 67),wherein C represents the amino acid cysteine, X represents a randomamino acid, m and n are numbers between 1 and 20 defining the length ofintervening polypeptide segments and I and o are numbers between 0 and20 defining the length of the flanking polypeptide segments; (c) thepolypeptide encoded by the nucleic acid is linked to the nucleic acid bythe phage; and (d) the polypeptides in the polypeptide library areattached to tris-(bromomethyl)benzene (TBMB) by three discrete covalentbonds between said TBMB and said polypeptide via said C amino acidresidues, and form at least one loop which comprises a sequence of twoor more amino acids subtended between two of said C amino acid residues,thus forming the one or more peptide ligand.
 2. The genetically encodedpolypeptide library of complexes of claim 1, wherein the polypeptidecomprises a sequence SEQ ID No. 1-5, 9, 10, or 36-63.